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Characterization of libid-based DNA delivery systems Mok, Kenneth W.C. 1998

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C H A R A C T E R I Z A T I O N O F LIPID-BASED DNA D E L I V E R Y SYSTEMS by K E N N E T H W. C. M O K B.Sc. (Hons.), Chemistry and Biochemistry, University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF BIOCHEMISTRY AND M O L E C U L A R BIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1998 © Kenneth W.C. Mok, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of BIOCHEMISTRY & MOLECULAR BIOLOGY The University of British Columbia Vancouver, Canada Date Augus t .25, 1998 DE-6 (2/88) ABSTRACT This thesis is focused on characterizing two lipid-based gene delivery systems: plasmid DNA-cationic lipid "complexes" and stabilized plasmid-lipid particles (SPLP). Complexes have utility for gene transfer in vitro whereas SPLP are designed for systemic gene therapy applications in vivo. In Chapter 2, the structural and fusogenic properties of complexes formed by mixing pCMV5 plasmid DNA with large unilamellar vesicles (LUVs) composed of the cationic lipid 7^-[2,3-(dioleyloxy)propyl]-7V,A ,^A^-trimethylammonium chloride (DOTMA) and l,2-dioleoyl-3-phosphatidylethanolamine (DOPE) or l,2-dioleoyl-3-phosphatidylcholine (DOPC) are examined and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between these cationic vesicles. Freeze fracture electron microscopy studies demonstrate association of cationic vesicles to form clusters at low pCMV5 content, 31 whereas macroscopic fused aggregates can be observed at higher plasmid levels. P NMR studies on the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 3 1 P NMR resonances, whereas T 1 the corresponding DOPC containing systems exhibit much broader "bilayer" P N M R spectra. In agreement with previous studies, the transfection potency of the DOPE containing systems is dramatically higher than for the DOPC containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. It is suggested that the P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of non-bilayer ii lipid structures, which may play a direct role in the fusion or.membrane destabilization events vital to transfection. In Chapter 3, the influence of variations in the lipid component of SPLP on plasmid trapping and transfection potency in vitro are characterized. It is shown that SPLP formed with different monovalent cationic lipids exhibit similar plasmid entrapment properties but different transfection potencies. The poly(ethylene glycol) (PEG) density in SPLP can substantially influence both SPLP formation and transfection. By decreasing the length of the fatty acyl component of the PEG-ceramide anchor from 20 to 14 to 8 carbons, or by using smaller PEG chains (PEG750, PEG2000 as compared with PEG5000X higher transfection levels were observed, consistent with a requirement for PEG removal in order for efficient transfection to occur. Further, it is shown that the primary factor limiting the transfection potency of SPLP is association and uptake into target cells. The final set of experiments in Chapter 4 was focused on characterizing the influence of the plasmid component in the formation of SPLP. It is shown that encapsulation efficiencies remain at 50 % or higher for (initial) plasmid-to-lipid ratios of up to 70 ug/pmol, allowing the proportion of lipid in empty vesicles following detergent dialysis to be significantly reduced compared to previous protocols. In addition, it is shown that the encapsulation efficiency is sensitive to the conformation of the plasmid employed, where higher encapsulation is observed for linearized plasmid as compared to plasmid in supercoiled or relaxed circular conformations. However, lower transfection potency for linearized plasmid was observed in SPLP and plasmid DNA-cationic lipid complexes. iii TABLE OF CONTENTS A B S T R A C T ii T A B L E OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix ABBREVIATIONS xii A C K N O W L E D G M E N T S xvii DEDICATION xviii CHAPTER 1: INTRODUCTION 1.1 Project overview: Lipids as DNA carriers 1 1.1.1 Methods in DNA delivery 1 1.1.2 Nucleic acid transfection techniques 2 1.2 Lipids 5 1.2.1 Chemistry and physics of lipids 5 1.2.1.1 Glycerophospholipids 8 1.2.1.2 Sphingolipids 8 1.2.1.3 Cholesterol 10 1.2.2 Structural behavior of lipids 10 1.2.2.1 Gel and liquid crystalline phase transition 10 1.2.2.2 Lipid polymorphism 12 1.2.3 Classification and preparation of liposomes 18 1.2.3.1 Multilamellar vesicles (MLVs) 18 1.2.3.2 Large unilamellar vesicles (LUVs) 18 1.2.3.3 Small unilamellar vesicles (SUVs) 20 1.2.3.4 Extrusion techniques 20 1.2.3.5 Micelles and detergent dialysis 21 1.2.4 Membrane Fusion 24 1.2.4.1 Fusion intermediate structures 24 1.2.4.2 Lipid mixing assay 28 1.3 Deoxyribonucleic acid (DNA) 31 1.3.1 Structural properties of DNA 31 iv 1.3.2 Plasmid DNA 35 1.4 Properties of liposomes for in vivo application 37 1.4.1 Properties of liposomes influencing circulation lifetime 37 1.4.2 Poly(ethylene glycol)-conjugated lipids 38 1.5 Cationic lipid-mediated DNA transfection 39 1.5.1 Diversity of cationic liposomes 39 1.5.2 Current models of DNA-cationic lipid complexes 44 1.5.3 Current hypotheses on mechanisms of lipid-based DNA delivery 48 1.6 D N A encapsulation 51 1.6.1 Approaches for encapsulating DNA in liposomes 51 1.6.2 Stabilized plasmid-lipid particles: construction and characterization 53 1.7 Thesis objectives 57 CHAPTER 2: STRUCTURAL AND FUSOGENIC PROPERTIES OF CATIONIC LIPOSOMES IN THE PRESENCE OF PLASMID DNA 2.1 Introduction 58 2.2 Materials and methods 60 2.2.1 Lipids and chemicals 60 2.2.2 Synthesis of D O T M A 61 2.2.3 Plasmid preparation 62 2.2.4 Preparation of large unilamellar vesicles (LUVs) 64 2.2.5 Lipid-mixing fusion assay 64 2.2.6 3 1 P N M R spectroscopy 66 2.2.7 Freeze fracture electron microscopy 67 2.2.8 Separation and quantification of DNA-cationic lipid complexes 68 2.2.9 In vitro DNA transfection on BHK cells 69 2.3 Results 71 2.3.1 D O T M A can stabilize DOPE into a bilayer organization 71 2.3.2 pCMV5 can trigger membrane fusion between DOTMA/DOPE LUVs.. . 73 2.3.3 Addition of pCMV5 to DOTMA/DOPE LUVs causes the formation of large lipid structures characterized by isotropic motional averaging.. 75 2.3.4 Addition of pCMV5 to DOTMA/DOPC (1:1) LUVs causes formation of large bilayer lipid structures 79 2.3.5 DOTMA/DOPE LUVs fuse with anionic vesicles and pCMV5 inhibits such fusion 83 2.3.6 Complexes formed by addition of plasmid DNA to cationic LUVs can be separated into two fractions by centrifugation 85 2.3.7 The transfection potency of pCMVpgal-DOTMA/DOPE (1:1) complexes correlate with the pellet fraction 88 2.4 Discussion 93 CHAPTER 3: STABILIZED-PLASMID LIPID PARTICLES: FACTORS INFLUENCING DNA ENTRAPMENT AND TRANSFECTION PROPERTIES 3.1 Introduction 98 3.2 Materials and methods 100 3.2.1 Lipids and chemicals 100 3.2.2 Plasmid 101 3.2.3 Preparation of plasmid DNA-cationic lipid complexes 101 3.2.4 Preparation of stabilized plasmid-lipid particles 101 3.2.5 Quantification of DNA entrapment and lipid recovery using anion exchange column chromatography 103 3.2.6 Purification of SPLP using sucrose density gradient centrifugation 103 3.2.7 Size determination of SPLP employing quasi-elastic light scattering 107 3.2.8 Transfection studies employing BHK cells 107 3.2.9 Cellular uptake studies of plasmid DNA 108 3.2.10 Southern blot analysis of delivered plasmid DNA 109 3.3 Results I l l 3.3.1 Influence of cationic lipid species on formation of SPLP I l l 3.3.2 Influence of the PEG polymer anchor on formation of SPLP 114 3.3.3 Influence of the PEG polymer anchor on transfection properties of SPLP 114 3.3.4 Influence of cationic lipid species on transfection properties of SPLP 120 3.3.5 Influence of PEG polymer length on formation and transfection properties of SPLP 120 3.3.6 Comparison of intracellular delivery of plasmid by SPLP and complexes 123 3.4 Discussion 127 CHAPTER 4: STABILIZED PLASMID-LIPID PARTICLES: INFLUENCE OF PLASMID CONFORMATION ON DNA ENTRAPMENT AND TRANSFECTION PROPERTIES vi 4.1 Introduction 132 4.2 Materials and methods 133 4.2.1 Lipids, chemicals and plasmid 133 4.2.2 Purification of plasmid of different conformations 133 4.2.3 Quantification of DNA entrapment by PicoGreen assay 134 4.3 Results 136 4.3.1 Influence of plasmid-to-lipid ratio on plasmid entrapment 136 4.3.2 Effects of plasmid conformation on entrapment 138 4.3.3 SPLP formed from supercoiled plasmid give the highest in vitro transfection 141 4.3.4 Stability of plasmid conformations in SPLP 141 4.4 Discussion 146 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS 5.1 Summary 149 5.2 Future Directions 152 REFERENCES 154 v i i LIST OF TABLES Table 1 Common methods of transfection 3 Table 2 Transition temperatures of various phospholipids composed of different acyl chain length, degree of saturation, and head group moiety 13 Table 3 Commercially available cationic lipid-based transfection reagents 43 Table 4 Methods of DNA entrapment in lipid systems 52 Table 5 Purification of SPLP for transfection 106 vii i LIST OF FIGURES Figure 1.1 Amphipathic nature of lipids and lipid movements in bilayer configuration 6 Figure 1.2 Schematic diagram of a biological membrane 7 Figure 1.3 Structures of common lipids in biological membrane 9 Figure 1.4 Gel to liquid-crystalline phase transition 11 Figure 1.5 Polymorphic phase behavior of lipids 15 Figure 1.6 Factors influencing the bilayer to hexagonal phase transition 17 Figure 1.7 Classification of liposomes 19 Figure 1.8 Schematic diagram of micelle formation 22 Figure 1.9 Schematic diagram of liposomes formation using detergent dialysis.... 23 Figure 1.10 Structures of detergents relevant to this thesis 25 Figure 1.11 Mechanisms of membrane fusion and intermediate structures 27 Figure 1.12 Schematic representation of lipid mixing assay 29 Figure 1.13 Structures of deoxyribonucleic acid 32 Figure 1.14 Conformations of plasmid DNA 34 Figure 1.15 Map of plasmid DNA used in this thesis 36 Figure 1.16 Structures of commonly used cationic lipids 41 Figure 1.17 Schematic representation of DNA-cationic lipid complexes 46 Figure 1.18 Mechanisms of lipid-based DNA delivery 49 Figure 1.19 Models of stabilized plasmid-lipid particles formation 56 ix Figure 2.1 Influence of D O T M A content in DOTMA/DOPE MLVs 72 Figure 2.2 Fusogenic behavior of DOTMA/DOPE vesicles in the presence of anions 74 Figure 2.3 3 1 P NMR of DOTMA/DOPE LUVs in the presence of anions 76 Figure 2.4 Freeze fracture E M of DOTMA/DOPE LUVs in the presence of plasmid DNA 78 Figure 2.5 Fusogenic behavior of DOTMA/DOPC vesicles in the presence of anions 80 Figure 2.6 3 'P NMR of DOTMA/DOPC LUVs in the presence of anions 81 Figure 2.7 Freeze fracture E M of DOTMA/DOPC LUVs in the presence of plasmid DNA 82 Figure 2.8 Fusogenic behavior of cationic vesicles with anionic model membrane vesicles 84 Figure 2.9 Fusogenic properties of plasmid DNA-cationic lipid complexes 86 Figure 2.10 Influence of charge ratios on the fractionation of DNA-cationic lipid complexes 87 Figure 2.11 3 1 P N M R of fractionated DNA-cationic lipid complexes 89 Figure 2.12 Transfection of complexes formed at different charge ratios 90 Figure 2.13 Transfection of the fractionated DNA-cationic lipid complexes 92 Figure 3.1 Purification of stabilized plasmid-lipid particles 105 Figure 3.2 Structures of cationic lipids used in this study 112 Figure 3.3 Influence of cationic lipids on plasmid encapsulation in SPLP 113 Figure 3.4 Structures of poly(ethylene glycol)-ceramides (PEG-Cer) used x in this study 115 Figure 3.5 Influence of ceramide anchors on plasmid entrapment properties in SPLP 116 Figure 3.6 Transfection properties of SPLP and plasmid DNA-cationic lipid complexes 118 Figure 3.7 Transfection properties of SPLP composed of different PEG-ceramide anchors 119 Figure 3.8 Comparison of the transfection properties of SPLP formed with different cationic lipids and DNA-cationic lipid complexes 121 Figure 3.9 Influence of PEG polymer lengths on plasmid entrapment in SPLP... 122 Figure 3.10 Influence of PEG polymer lengths on transfection properties of SPLP 124 Figure 3.11 Southern blot analysis of plasmid DNA delivered by SPLP and plasmid DNA-cationic lipid complexes in BHK cells 126 Figure 4.1 Influence of total lipid content on DNA entrapment 137 Figure 4.2 Influence of plasmid DNA content on DNA entrapment 139 Figure 4.3 Agarose gel electrophoretic patterns of pCMVPgal plasmid of different conformations 140 Figure 4.4 Plasmid entrapment properties of SPLP containing pCMVPgal of different conformations 142 Figure 4.5 Comparison of the transfection ability of DNA-cationic lipid complexes and SPLP formed with different plasmid conformations.... 143 Figure 4.6 Characterization of the influence of encapsulation on plasmid ^ conformation by gel electrophoresis 145 xi ABBREVIATIONS A adenine p-gal P-galactosidase B H K baby hamster kidney bp base pair BSA bovine serum albumin C cytosine C A T chloramphenicol acetyltransferase 1 4 C - C H E 14C-labeled cholesteryl hexadecyl ether C D A B cetyldimethylethylammonium bromide cmc critical micellar concentration Choi cholesterol CL cardiolipin CPRG chlorophenol red galactopyranoside C T A B cetyltrimethylammonium bromide C T A C cetyltrimethylammonium chloride C M V cytomegalovirus DC-CHOL S-yS-t/V-CTV^-dimethylaminoethy^carbamoylj-cholesterol DDAB dimethyldioctadecylammonium bromide D M E M Dulbecco's Modified Eagle Medium D E A E diethylaminoethyl DMRIE A^-[2,3-(dimyristyloxy)propyl]-A^,7V-dimethyl-A^-hydroxyethylammonium bromide xii DNA deoxyribonucleic acid DNase deoxyribonuclease I DODAB iV,A^-dioleyl-iV",iV"-dimethylanimonium bromide D O D A C A^,A^-dioleyl-iV,iV-dimethylammonium chloride D O D M A - A N A^-[2,3-(dioleyloxy)propyl]-A ,^A^-dimethyl-A^-cyanomethylammonium chloride DOPC 1,2-dioleoyl-3-phosphatidylcholine DOPE l,2-dioleoyl-3-phosphatidylethanolamine DOPS 1,2-dioleoyl-3-phosphatidylserine DOGS dioctadecylamidoglycylspermine DOSPA 2,3-dioleyloxy-A^-[2-(sperminecarboxamido)ethyl]-7V,7V-dimethyl-1 -propanaminium trifluoroacetate DOSPER l,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid tetraacetate DOTAP 1,2-dioleoyloxy-3-(trimethylammonio)propane D O T M A A^-[2,3-(dioleyloxy)propyl]-A ,^A ,^A^-trimethylammonium chloride DSDAC N-distearyl-Af A^imethylammonium chloride DTAB dodecyltrimethylammonium bromide E D T A ethylenediaminetetraacetic acid E M electron microscopy EPA egg phosphatidic acid EPC egg phosphatidylcholine FBS fetal bovine serum G guanine Xlll 3 H tritium labeled HBS HEPES buffered saline (150 mM NaCl, 20 mM HEPES, pH 7.4) Hn hexagonal phase HEPES 7Ar-(2-hydroxyethyl)piperazine-7Y'-2-ethanesulfonic acid IMI interlamellar micelle intermediate or inverted micellar intermediate kb kilobase L a liquid crystalline state Lp gel state LUVs large unilamellar vesicles MLVs multilamellar vesicles M W molecular weight NBD-PE JV-(7-nitro-2,l ,3 -benzoxadiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine OGP n-octyl-/?-D-glucopyranoside PBS phosphate buffered saline QELS quasielastic light scattering RES reticuloendothelial system Rh-PE 7V-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-s«-phosphatidylethanolamine PA phosphatidic acid PC phosphatidylcholine pCMV5 plasmid containing cytomegalovirus promoter (5000 bp) pCMVPgal plasmid containing cytomegalovirus promoter and p-gal gene (7164 bp) xiv p C M V C A T plasmid containing cytomegalovirus promoter and C A T gene (4421 bp) PE phosphatidylethanolamine PEG-CerCg 1 -0-(2'-(©-methoxypolyethyleneglycol(2ooo))succinoyl)-2-A^-octanoylsphingosine PEG-CerC 14 1 -0-(2' -(oo-methoxypolyethyleneglycol(2ooo))succinoyl)-2-7Y-myristoylsphingosine PEG-CerC2o l-0-(2'-(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-A^-arachidoylsphingosine PEG750 1 -0-(2' -(co-methoxypolyethyleneglycol(750))succinoyl)-2-7Y-myristoylsphingosine PEG5000 l-0-(2'-(©-methoxypolyethyleneglycol(5ooo))succinoyl)-2-7V-myristoylsphingosine PG phosphatidylglycerol PI phosphatidylinositol PL phospholipid 3 1 P NMR phosphorus-31 nuclear magnetic resonance PS phosphatidylserine SA stearylamine SM sphingomyelin SPLP stabilized plasmid-lipid particles T thymine T D A AT,7v',7V',7vr'-tetramethyl-A y^/V'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide T E Tris-EDTA buffer T H transition temperature T M C trans-monolayer contact TMTPS Af,Af',Af'',Af'"-tetramethyl-Afr/V',iV'',Af'"-tetrapalmitylspermine T R C - T E M time-resolved cryo-transmission electron microscopy Tris tris(hydroxymethyl)aminomethane Triton X-100 ^-octylphenoxypolyethoxyethanol T T A B tetradecyltrimethylammonium bromide % AF/AFmax percent change in fluorescence (defined in Chapter 2) xvi A C K N O W L E D G M E N T S I would like to thank everyone in the Cullis lab for their support and jokes, especially to Drs. David Fenske, Kim Wong, and Pat Tarn for their instruction and advice on 3 1 P NMR, freeze fracture electron microscopy, and plasmid preparation techniques, respectively. I would also like to acknowledge Dr. Barb Mui for teaching me the fluorescence fusion assay. Special thanks to Lome Palmer for sharing his unique procedure and advice on the development of SPLP systems, and Angel Lam for her time in teaching cell culture techniques, and her collaboration and contribution to the results of plasmid cellular uptake data to this thesis. In addition, I would like to thank Dr. Peter Scherrer of Inex Pharmaceuticals Corporation (Burnaby, BC) for sharing his advice in developing SPLP. I would also like to acknowledge Dr. S. Ansell of Inex for the generous supply of different cationic lipids, Z. Wang of Inex for supplying various PEG-Cer, and A. Annuar of Inex for supplying H-labeled plasmid. In addition, I must acknowledge the 5 years of financial support from the Medical Research Council of Canada. Finally, I must thank my supervisory committee members, Dr. Michael Hope and Dr. Ross MacGillivray for their time and suggestions throughout my Ph.D program. I would like to also thank Dr. D. Fenske for reading this thesis. Most importantly, I must express my gratitude to Dr. Pieter Cullis for his continuous encouragement, advice, and ideas over the past six years, in addition to an outstanding and enjoyable laboratory. xvii To my parents and brothers, and for my special friend, J-K xviii CHAPTER 1 INTRODUCTION 1.1 PROJECT OVERVIEW: LIPIDS AS DNA CARRIERS The development of delivery systems for introducing genetic materials into cells has been a focus of research for more than 40 years, but it is only during the past decade that lipids have been used as DNA delivery systems. In this section, an overview of different gene delivery methods and their associated problems are presented. 1.1.1 Methods for DNA delivery Currently there are two different approaches in gene delivery. The first approach utilizes viruses as vectors to introduce the gene into target cells. This approach involves the construction of viral particles that lack pathogenic functions, are incapable of replication, contain a therapeutic gene within the viral genome, and can deliver this gene to cells by the process of infection. Some of the common viral vectors include retroviruses (Miller, 1990; Dunckley and Dickson, 1994), adenovirus (Berkner, 1988; Stratford-Perricaudet and Perricaudet, 1994), adeno-associated virus (McLaughlin et al., 1988; Muzyczka, 1992), herpes simplex virus (Andersen et al., 1992; Glorioso et al., 1994), and vaccinia virus (Morrison et al., 1989; Morrison et al., 1991). Limitations of viral vectors include the small size of the gene that can be introduced into the viral genome, as well as viral immunogenicity which limits the utility of repeat injections. In addition, the safety of using viral vectors is a major concern due to the possibility of recombination events l leading to the generation of an infection competent virus. Viruses such as retroviruses can only infect dividing cells. Furthermore with the possible exception of adeno-associated virus, which preferentially integrates into a specific region of the host chromosome, viral vectors insert heterologous DNA randomly into the host genome and thus may activate a proto-oncogene. These problems with viral vectors highlight the need for non-viral approaches. During the past three decades, non-viral methods for gene delivery have rapidly evolved (Table 1). This approach involves delivering plasmid DNA containing the gene of interest by means of physical techniques such as microinjection, electroporation, and biolistic particle bombardment; or by incubating the DNA with chemicals such as D E A E -dextran, calcium phosphate (CaPO^), polybrene, and cationic lipids. 1.1.2 Nucleic acid transfection techniques The two earliest nucleic acid transfection methods, DEAE-dextran (Vaheri and Pagano, 1965) and CaP04 co-precipitation (Graham and Van der Eb, 1973), are still popular methods for DNA transfection. These methods rely on having the plasmid DNA adsorbed on the surface of dextran or co-precipitated with CaPC^, with subsequent uptake of the resulting complexes by the transfecting cells. However, these methods often result in inconsistent and low transfection efficiencies. In the 1980s, physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Neumann et al., 1982) and biolistic particle delivery (Ye et al., 1990; Yang et al., 1994) were introduced. Although these methods could yield higher transfection efficiencies in some cell lines, none could be used widely for all cell types. In addition, inconsistent 2 J 4> o\ oo CS OH <L) •a > o oo ON CJ & U 00 OS N z ! oo ON CJ c _6fl O N O N "c3 CJ oo a <D C3 -> >> 1) 01 ca ea 2 2" d cj +-» yi on on o o o o .3 HJ .3 c/5 J J - S & O 3 § =3 OO &0 H CJ —' c B. ° 13 g -g -a S ^ | § e u 3 * in O CJ .£ 45 *r CM g * o J3 — , <*> o -S3 "u H- -tn VI cj <8 § -a y o <u O I oo E3 I"3 o CJ p . oo I C o z ^3 a. 4> <-> ro JT3 ^ B E 1 " 00 p , O IS '3 -a & Ir.« O cj • t/3 on > •a _> <4H G u .9 *>H OO O u cj > "c l o u M is of cj oo <£ o +2 'G 43 T J a g •» g <*> S. o 2 X o o c c CJ C o CS PH CD > o O CJ rv -3 .S U H H — 'cn 3 cj o «M •£ -8 ° o o '!§ & . H ^ 2 cj o o SH CH c4 o •a cu CU "3 Q .3 CJ O < Z a z Q CJ <% Z ° d cj - 3 05 - H cj Z o Q & o t H D . .3 o CH z Q < CJ Z o s « Z o Q JP o < Z Q CLP a 4H O <L> to! .3 & CJ 60 = o T3 a o on nl t3 CJ C M O G SP >H - C CU CH o u a, CJ t H HO a CJ a 4H O CJ .3 & CJ 60 = o T3 ci a o O CJ 2 & CJ 00 SH CS u 3 o C O H 6 3 T H x <u •a i a on ion ne sp O CH u ea ne a, por bre ea • ~ o '5 U o i . _© Mic Elec PH Cat •3 S . _ cu ea .a S? S Si ca 3 transfection results, the inability to transfect certain cells, cell toxicity, and the cost of expensive equipment reduce the utility of these transfection techniques. The current era began with Feigner et al. (1987), who developed a transfection technique using cationic lipid as an agent for gene transfer. Cationic lipid-mediated transfection is simple, economical and gives high transfection efficiencies in most cell types. Today, several cationic lipid-based transfection reagents are commercially available (see section 1.5). Table 1 summarizes the versatility and mechanism of common transfection techniques. This thesis characterizes two lipid-based DNA delivery systems: the plasmid DNA-cationic lipid complexes commonly used for lipid-based gene delivery, and the stabilized plasmid-lipid particles (SPLP) designed as an in vivo gene delivery system. In order to understand the characteristics of both systems, the properties of lipids and plasmid DNA are first introduced. This is followed by a section describing the properties of liposomes in in vivo applications. The final two sections of the introduction describe the current knowledge of cationic lipid-mediated plasmid DNA transfection and plasmid DNA encapsulation, respectively. 4 1.2 LIPIDS Lipids are important molecules in biological systems. Some are used for energy storage as fats, while the other cellular lipids are assembled into lipid membranes, which separate the cellular compartments from the external environment and mediate signal transduction processes for cellular regulation. Membrane lipid molecules are "amphipathic", possessing hydrophilic and hydrophobic regions in the same molecule (Figure 1.1). This amphipathic nature of lipid molecules allows bilayer forming lipids to self-assemble into bilayer membranes, in which the lipid head groups are oriented towards the aqueous environment, while the hydrophobic tails are facing each other internally. Biological membranes are composed of lipids, proteins, and carbohydrate with virtually all the carbohydrate covalently bound to proteins or lipids (Figure 1.2). They form selective barriers that determine the permeability of particular substances. As described by Singer and Nicholson (1972), the "fluid-mosaic" nature of the lipid bilayers allows for considerable molecular motion, both lateral and rotational, in the lipid matrix, and provides an appropriate milieu for the functions of membrane proteins. 1.2.1 Chemistry and physics of lipids Lipids can be classified into different groups depending upon their structures and functions. Lipids used for energy storage and energy metabolism consist of fatty acids and triacylglycerols. The lipids which comprise biological membranes can be classified into three groups: glycerophospholipids, sphingolipids, and sterols. In the following sections, only lipids relevant to the investigations of this thesis are discussed. 5 Figure 1.1 Amphipathic nature of lipids and lipid movements in bilayer configuration 6 Figure 1.2 Schematic diagram of a biological membrane Fluid mosaic model of the biological membrane with intrinsic proteins embedded in the lipid bilayer and extrinsic proteins attached on the membrane surface. The carbohydrate moieties on lipids or proteins face the exterior surface of the cell (<§•)• 7 1.2.1.1 Glycerophospholipids Glycerophospholipids (or phosphoglycerides) are the major class of naturally occurring phospholipids, and contain a glycerol-based backbone and phosphate-containing head groups. These lipids which are the predominant lipid species in eukaryotes, include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and cardiolipin (CL). The structures of different phospholipids are depicted in Figure 1.3. Most properties of different phospholipids are determined by the head group. For example, PS, PG, PI and PA are anionic, while PC and PE are zwitterionic. Additional diversity of phospholipids can result from the length (from 16 to 24 hydrocarbons) and the degree of unsaturation in the two acyl chains. Typically, the most common saturated fatty acids are palmitic (16 hydrocarbons, 0 double bond; conventionally designated as 16:0) and stearic acid (18:0), whereas the most abundant unsaturated fatty acids are oleic (18:1) and linoleic acid (18:2) (Figure 1.3). Mammalian phospholipids usually possess two different fatty acids, with a saturated chain at the carbon 1 (C-l) position and an unsaturated chain at the C-2 position of the glycerol backbone. 1.2.1.2 Sphingolipids A second major class of membrane lipids derive from the long-chain amino alcohol sphingosine (Figure 1.3). Linkage of a fatty acid via an amide bond to the amino group of sphingosine, yields a class of sphingolipid referred to as ceramide. Attachment of head groups to the hydroxyl group at C- l leads to common membrane lipids, such as sphingomyelin (SM) (Figure 1.3) and the glycolipids. 8 Figure 1.3 Structures of common lipids in biological membrane Phospholipid (PL) R i 0 = P - 0 4 H 1 H X - C - C H A i o W W o I Headgroup ^ > Glycerol Cholesterol (Choi) Backbone : £ > Acyl chain Phosphatidylcholine (PC) R-CH 2CH 2N(CH 3) 3 Phosphatidylethanolamine (PE) R-CH 2CH 2NH 3 Phosphatidic acid (PA) R-H Phosphatidylserine (PS) H + R C H 2 — C - N H 3 COO" Phosphatidylglycerol (PG) R-CH 2CH(OH)CH 2OH Phosphatidylinositol (PI) OH OH Some naturally occurring fatty acids Laurie (12:0) Myristic (14:0) Palmitic (16:0) Stearic (18:0) CH 3 (CH 2 ) , 0 COOH CH 3 (CH 2 ) , 2 COOH CH 3 (CH 2 ) 1 4 COOH CH 3 (CH 2 ) , 6 COOH Palmitoleic (16:1,A ) CH 3(CH 2) 5CH=CH<CH 2) 7COOH Oleic (18:1, A9) CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH Linoleic (18:2, A9'12) CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH Linolenic(18:3 ,A 9 ' 1 2 ' 1 5 ) CH 3 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH Sphingolipid R-NH OR' R = H; R' = H R = COR"; R' = H R = COR"; R' = phosphocholine (R" = hydrocarbon) Sphingosine Ceramide (Cer) Sphingomyelin (SM) 9 1.2.1.3 Cholesterol Cholesterol (Choi) is the most abundant sterol and the major neutral lipid component of eukaryotic membranes. It is a weakly amphipathic substance, due to the 3p-hydroxyl group that is oriented towards the lipid-water interface, and situated next to the carbonyl groups of the acyl chains of phospholipids when inserted in the bilayer membrane. The fused cyclohexane rings make cholesterol a bulky, rigid structure embedding in the hydrophobic interior of the bilayer membrane (Figure 1.3). Cholesterol increases the order (decreases the fluidity) of a membrane. 1.2.2 Structural behavior of lipids The structural behavior of lipids is not only dependent upon the types of lipid, but also upon the surrounding environment. Lipids adopt particular organizations or phases when hydrated. While the most common phase is the. lipid bilayer, a variety of non-bilayer phases, such as the hexagonal Hn or isotropic phases, have been characterized. The propensity of lipids to adopt different phases in response to external variables (such as pH, temperature, or the presence of ions) is referred to as lipid polymorphism. In addition, phospholipids in the bilayer phase can be in two different states, the gel or liquid crystalline state, depending on the temperature. 1.2.2.1 Gel and liquid crystalline phase transition Phospholipids can exist in either a frozen "gel" state (Lp) or a fluid "liquid crystalline" state (L a) depending on the temperature (Figure 1.4A). This transition temperature (TH) is specific for each lipid species. Below T H , the carbon-carbon bonds of the acyl chains tend 10 Figure 1.4 Gel to liquid-crystalline phase transition (A) The transition between the gel and liquid crystalline states in a bilayer. The acyl chains of lipids arranging in a gel state are tilted and ordered. (B) A fatty acid undergoes a transition from the all-trans state to a gauche isomer with a "kink" formation in the acyl chain. 11 to be extended into an all-trans conformation, resulting in rigid or ordered acyl chains. This phase is referred to as the gel state ( L p ) . As the temperature approaches T H , gauche isomers start to form and "kinks" in the acyl chains appear (Figure 1.4B). When the temperature is above T H , the average number of gauche isomers per acyl chain increases, resulting in a shortening of the acyl chains and an increase in the packing distance between individual lipid molecules. The lipids are now in the liquid crystalline (L a ) state where rapid anisotropic motions occur, including lateral diffusion of lipid molecules in the plane of the membrane. The T H is dependent on both the properties of the acyl chains and lipid head group (Table 2). Higher T H occurs if the acyl chain length is increased for a given lipid species. Also, lipids with saturated acyl chains have higher T H than the unsaturated ones. Charge repulsion of lipid head groups can increase lateral diffusion of lipid molecules and favors L a state. Thus, charged lipids tend to have lower T H - Although lipids arranged in the gel state can be obtained in vitro, there is no evidence of gel state lipids being present in eukaryotic membranes at physiological temperatures (Cullis and Hope, 1985). 1.2.2.2 Lipid Polymorphism Liquid crystalline lipids can be arranged into different lipid polymorphic phases upon hydration. These include the "lamellar" or "bilayer" phase, the "hexagonal" phase (Hn) and other non-bilayer phases grouped as the "cubic" phases. Non-bilayer forming lipids can be found and isolated from all biological membranes. Various factors can influence lipid polymorphism. These include intrinsic factors, such as the nature of the lipid head group or acyl chains, and extrinsic factors, such as 12 Table 2 Transition temperatures of various phospholipids composed of different acyl chain length, degree of saturation, and head group moiety Lipid species Transition temperature (°C) dilauroyl PC (12:0, 12:0) -1 dimyristoyl PC (14:0, 14:0) 24 dipalmitoyl PC (16:0, 16:0) 41 distearoyl PC (18:0, 18:0) 55 stearoyl, oleoyl PC (18:0, 18:1) 6 stearoyl, linoleoyl PC (18:0, 18:2) -13 dipalmitoyl PA (16:0, 16:0) 67 dipalmitoyl PE (16:0, 16:0) 63 dipalmitoyl PS (16:0, 16:0) 55 dipalmitoyl PG (16:0, 16:0) 41 13 hydration, pH, temperature, ionic strength, or the presence of other molecules including ions, lipids, or proteins. Lipid polymorphism can be attributed to the molecular shape of the lipid molecule, which is dependent upon the relative size of the three-dimensional volume occupied by the hydrophilic head group and the hydrophobic tail (Israelachvili et al., 1980; Cullis et al., 1986). The polymorphic properties of different lipids have been extensively reviewed elsewhere (Cullis and de Kruijff, 1979; Gruner et al., 1985; Lindblom and Rilfors, 1989; Seddon, 1990). Bilayer forming lipids have a cylindrical molecular shape arranged in a planar organization (Figure 1.5). Pure lipid systems demonstrating this packing behavior include those formed with PC, PS (pH > 4.0), PI, PG, PA (pH > 3.0), C L , and SM. Some lipids, such as unsaturated PE, PS (pH < 4.0), and PA (pH < 3.0), have a small head group volume relative to the acyl chains, and can be thought to have a "cone" shaped arrangement. These lipids tends to form the Hn phase, in which the lipids are in a tubular arrangement with the hydrophilic head groups facing inward interacting with aqueous channels, and the hydrophobic tails interacting with the acyl chains of other lipid molecules (Figure 1.5). Lipids that can adopt the Hn phase usually represent at least 30 mol % of the total lipids in biological membranes. In contrast, lipids with large head groups compared to the tail portion can be thought to possess an "inverted cone" geometry resulting in micelle formation (Figure 1.5). These include different detergents, and lysophospholipids (which contain only one acyl chain). The phase preference of a given lipid can be modulated by adding different lipids, adding counter-charged ions, or manipulating the pH or temperature of the system. As demonstrated in earlier works, addition of 20 to 50 mol % PC or PS (pH > 4.0) into pure 14 o CL O C O c o C D CO X 0) X E CL CL O 09 T 3 < - I LU Z3 CL o < LU x 8 0 5 CD c o O "O CD CD > CO o •a O c o O 4> -3 s-o > CU Vi cs -S C o E >? o OH TO "CD O a) _ro S ^yP> w— < s s i y © c o c o c o ® co c o O) co X CD I C O a Q. oo •g ~ " CO o "£ •C CD CL OJ CO t. O CD o Q CD c -£= Q) O >, I f CL SZ CO CL O CO A o CD .-= JE >, J2 ro >*.c: •D CL 4= 00 CO O .c sz Q-Q_ 00 o _ ^ O A CD X i s . _g>-g a. >>'o 7; -g < .2 'ro o "S .c ^ ro C L S O « CO 2 sz £ CL 0- 00 o CD I _ r o o c ro _C -*—• JD >. T3 CL 00 o JZ + CN ro ro 1 O !2 c < Q..2 i s T3 ro ro CL O 00 CO 2 g " c: < CD O S >• co "D ™ -E -P ro 00 co 00 a- ^ 15 PE systems can stabilize the Hn phase into bilayer organization (Cullis et al., 1985; Seddon, 1990). Mixing micelle-forming species such as detergents with PE can also result in bilayer organization (Madden and Cullis, 1982). Furthermore, the addition of divalent cations such as C a 2 + to mixed anionic lipid PE/PS systems can change the polymorphic behavior from bilayer to Hn phase. This occurs because the binding of C a 2 + to PS can laterally segregate PS in the fluid bilayer, allowing PE to adopt the Hn phase (Cullis and de Kruijff, 1979). Similar behavior can be observed if the pH of the PE/PS system is decreased to < 3.0. This is due to protonation of PS, and thus a reduction in the effective volume of the PS head group (Tilcock et al., 1988). The promotion of Hn phase by increasing temperature and decreasing hydration have also been studied by 3 1 P N M R (Tilcock et al., 1982; Tilcock and Cullis, 1987). A summary of factors that influence the inter-conversion of bilayer to Hn phase is shown in Figure 1.6. The polymorphic behavior of many lipid systems has been observed using phosphorus (31P) nuclear magnetic resonance (NMR) and freeze fracture electron microscopy (EM). In 3 1 P NMR, a characteristic bilayer spectrum is observed which, exhibits a high field peak and low field shoulder separated by about 40 ppm. In contrast, the Hn phase exhibits the reverse asymmetry with a spectral width about half that of the bilayer signal. Small systems such as micelles and vesicles, and systems having rapid motional properties such as the cubic phases exhibit isotropic motional averaging in the 3 1 P N M R signal (Gruner et al., 1985; Tilcock and Cullis, 1987). Cubic phases are very complex (Lindblom and Rilfors, 1989), and while neither cubic nor Hn phases are observed in vivo, the intermediate structures involved in bilayer to non-bilayer transitions are thought to play a role in membrane fusion (Siegel, 1993). 16 Figure 1.6 Factors influencing the bilayer to hexagonal phase transition 17 1.2.3 Classification and preparation of liposomes Model membrane systems, such as monolayers, planar bilayers, and liposomes, are convenient models of biological membranes for studying the physical properties and functional roles of lipid components. Because liposomes exhibit excellent biocompatibility properties and may be used to entrap many drugs, they are also utilized as in vivo drug carriers. Liposomes may contain more than one lipid bilayer, and can be classified into three types. 1.2.3.1 Multilamellar vesicles (MLVs) The first type of liposomes that can be easily formed upon hydration of a lipid film are the multilamellar vesicles (MLVs) (Bangham et al., 1965). These vesicles are usually composed of concentric layers of bilayers and are heterogenous in size (0.5 - 10 urn) (Figure 1.7). MLVs have low trapping volumes (0.5 ul/umol), and have unequal transbilayer distributions of solutes. Equilibration of solute can be achieved by subjecting MLVs to freeze-thaw cycles (Mayer et al., 1985). MLVs are useful for studying the structural and motional properties of lipids. Because MLVs are relatively large, they tumble slowly on the NMR timescale, and the spectra observed reflect the local motions experienced by the lipid molecules. MLVs are not widely used as drug delivery vehicles due to their low trapping volumes and their large size, which leads to rapid clearance. 1.2.3.2 Large unilamellar vesicles (LUVs) Large unilamellar vesicles (LUVs) are the most commonly used model membrane systems for carrying drugs. LUVs possess a single bilayer and have sizes ranging from 50 18 Figure 1.7 Classification of liposomes Schematic representation and freeze fracture electron micrographs of (A) M L V s , (B) L U V s , and (C) S U V s are shown. The bar in the E M picture represents 200 nm. 19 to 200 nm (Figure 1.7). The smaller size of LUVs relative to MLVs results in longer circulation lifetimes in vivo (Juliano and Stamp, 1975). LUVs can be formed by several methods. These include reverse phase evaporation (Szoka and Papahadjopoulos, 1978), detergent dialysis (Mimms et al., 1981), injection of lipids containing organic solvents into aqueous buffer and the subsequent removal of the organic solvents through dialysis or gel filtration (Szoka and Papahadjopoulos, 1980), and extrusion techniques (Szoka and Papahadjopoulos, 1980; Mayer et al., 1986). Extrusion through polycarbonate filters with well defined pore size is the most convenient method for producing LUVs. 1.2.3.3 Small unilamellar vesicles (SUVs) Small unilamellar vesicles (SUVs) are single bilayer vesicles with diameters of about 25 to 50 nm (Figure 1.7). These vesicles are formed by subjecting MLVs to sonication (Huang, 1969) or by passage through a French press (Barenholzt et al., 1979). The small radii of curvature in SUVs give rise to rapid tumbling motional properties and result in an isotropic 3 , P N M R signal (Cullis et al., 1985). The trapped volume of SUVs is too small for them to be of major utility as drug delivery vehicles. 1.2.3.4 Extrusion techniques Extrusion is the most efficient method for forming homogenous LUVs (Szoka and Papahadjopoulos, 1980). This technique involves repeatedly forcing a lipid dispersion through two polycarbonate membrane filters (with pore sizes ranging from 50 to 400 nm) under pressure (300 - 400 psi). The size of the resulting vesicles is dependent on the pore size of the filters. Mayer et al. (1986) have demonstrated that passing MLVs ten times 20 through two stacked filters of 100 nm pore size results in a uniform distribution of 100 nm diameter vesicles. 1.2.3.5 Micelles and detergent dialysis Detergents, also known as surfactants, are amphiphiles that migrate to the air-water interface, or which form micelles, when placed in aqueous solution. They are often used in the isolation and study of integral membrane proteins, for reconstituting membrane proteins into liposomal systems (Madden and Cullis, 1984), and in liposome preparation (Szoka and Papahadjopoulos, 1980; Lasch et al., 1983; Kiwada et al., 1985; Almog et al., 1986). Similar to lipids, detergents have a hydrophobic tail and a hydrophilic head group (Figure 1.8). At low concentrations in aqueous solution, detergents exist as monomers dispersed in solution. When the concentration increases to a specific level, referred to as the critical micellar concentration (cmc), "micelles" or clusters of detergent molecules are formed (Figure 1.8). The cmc is a physical characteristic of each detergent. Most detergents have cmc values within a narrow range; however, detergents containing bulky rigid, hydrophobic portions, such as bile acids, can have a broad cmc range (Kratohvil, 1986). Micelles are dynamic structures: detergent monomers present in one micelle can constantly exchange with other monomers present in the bulk solution or in other micelles (Lindman and Wennerstrom, 1980). In order to form liposomes employing detergents, the lipid is first dissolved in detergent micelles and the detergent is then removed by dialysis. In some cases, the size of liposomes can be regulated by controlling the rate of detergent dialysis (Figure 1.9). Detergents can be classified by the charge or nature of the head group or by the chemical 21 Figure 1.8 Schematic diagram of micelle formation 22 Figure 1.9 Schematic diagram of liposomes formation using detergent dialysis 23 nature of the tail. The head groups may be anionic, cationic, zwitterionic, or non-ionic. The tail portion of the detergents can contain straight or branched hydrocarbons, or a steroid-based structure. Detergents most suitable for dialysis are those with high cmc values (> 1 mM) and small micelle size (Dencher and Heyn, 1978; Furth, 1980). Structures of the detergents used in this thesis are shown in Figure 1.10. 1.2.4 Membrane fusion Membrane fusion is a common biological event. Some representative examples include (1) the exocytotic process by which intracellular vesicles fuse with the plasma membrane, (2) the release of neurotransmitters such as acetylcholine from the pre-synaptic axon in the synaptic cleft in response to elevated C a 2 + levels, (3) the fusion between a spermatid with an oocyte during fertilization, and (4) the process by which viruses infect host cells. In this section, the current mechanism of membrane fusion and the intermediate structures involved are discussed. 1.2.4.1 Fusion intermediate structures During the past few decades, tremendous efforts have been devoted to understanding the role of non-bilayer lipids in biological membrane. A variety of physical techniques have allowed detection of lipid polymorphic structures in different lipid systems and conditions. These include 3 1 P NMR (Cullis and de Kruijff, 1979; Tilcock et al., 1984; Cullis et al., 1985), 2 H NMR (Seelig and Seelig, 1977; Bloom et al., 1991), freeze fracture E M (Verkleij, 1984; Cullis et al., 1985; Lindblom and Rilfors, 1989), time-24 Figure 1.10 Structures of detergents relevant to this thesis n-octyl-6-D-glucopyranoside (OGP) CH 2 OH HO O 0—(CH 2 ) 7 CH ; Cmc (mM)a non-ionic 19-25 )H Cetyltrimethylammonium bromide (CTAB) cationic 1.0 Br + N(CH 3) 3 Sodium dodecyl sulfate (SDS) anionic 1 - 2 O 04-0-O Na+ Triton X-100 non-ionic 0.29 / ^ 0 ( C H 2 C H 2 0 ) n H n = 9-10 Cmc values are at room temperature and 0.1 to 0.2 M Na+ 25 resolved cryo-transmission electron microscopy (TRC-TEM) (Siegel et al., 1989; Talmon et al., 1990; Siegel et al , 1994; Siegel and Epand, 1997), X-ray diffraction (for reviews, see Gruner et al., 1985; Seddon, 1990), differential scanning calorimetry (Cullis and deKruijff, 1979; Wu et al., 1982), and infrared spectroscopy (Mantsch et al., 1981). The results of many P NMR, freeze fracture E M , and T R C - T E M studies suggest that factors which promote Hn phase can also induce membrane fusion in liposomal systems (Cullis and de Kruijff, 1979; Verkleij, 1984; Cullis et al., 1986; Siegel and Epand, 1997). A variety of mechanisms of membrane fusion have been proposed (Papahadjopoulos et al., 1990). Three leading models of membrane fusion are summarized below. The first model involves the formation of "interlamellar micellar intermediates" (IMI), sometimes referred to as "inverted micellar intermediates", in which inverted micellar structures are formed between the two closely apposed bilayers in the fusion process (Figure 1.11 A). Evidence for these structures includes the presence of an isotropic 3 1 P NMR signal together with structures referred to as "lipidic particles" in the fractured surfaces of fused vesicles in freeze fracture E M (Cullis and de Kruijff, 1979; Verkleij, 1984; Cullis et al., 1986). It has been proposed that non-bilayer lipids, such as PE, favor the formation of these intermediate structures. The second model, sometimes called the "stalk-pore" model, involves a "stalk" intermediate (Markin et al., 1984; Chernomordik et al., 1985), which is formed by coalescence of the outer monolayers of the apposing membranes (Figure 1.1 IB). This semi-toroidal lipid structure is then expanded radially becoming a trans-monolayer contact (TMC). The T M C continue to radially expand to form a single bilayer diaphragm 26 on CU 3 +J u 3 la SO CU C S • aa T 3 CU a 3 • 3 i-H es CU ia 3 bD a ,© a CU 3 cs S-- D a cu a «*a O v> E on '3 eS .3 u ro E d) c "55 o E « o +•» c ©===535 (0 CO ( K c r a ) —> o-=-r==o T3 < -a > . - S J= E.E c .2 „ w c 3 o u . a 27 between the two apposed vesicles, termed a "hemifusion intermediate", which is then rapidly ruptured to yield a fusion pore (Chernomordik and Zimmerberg, 1995). The "modified stalk model" proposed by Siegel (1993) is an extension of the "stalk-pore" model. It also involves the initial formation of stalk and T M C intermediates, but the TMCs do not radially expand to form a "hemifusion intermediate". Instead, these TMCs mediate membrane fusion by having the diaphragm rupture locally in the T M C (Figure 1.1 IC). Evidence for this model comes mainly from different intermediates observed in T R C - T E M at various temperature below and above T H - (Siegel, 1993; Siegel et al., 1994; Siegel and Epand, 1997). By comparing the free energies associated with both intermediate structures, Siegel (1993) has shown that the stalk intermediate is an energetically favored structure. 1.2.4.2 Lipid mixing assay One of the common approaches in monitoring membrane fusion is to use a lipid mixing assay, sometimes referred to as a "resonance energy transfer" (RET) assay (Figure 1.12). This assay involves using two fluorescently labeled conjugated lipid probes, or fluorophores, with A^-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1,2-dioleoyl-.s«-phosphatidylethanolamine (NBD-PE) as the fluorescence donor and 7V-(lissamine rhodamine B sulfonyl)-l,2-dioleoyl-sn-phosphatidylethanolamine (Rh-PE) as the fluorescence acceptor. The fluorophores have been shown to be non-exchangeable between vesicles, even during membrane aggregation (Struck et al., 1981). In practice, the two fluorophores are incorporated in the same population of vesicles, and are then mixed with a population of non-labeled vesicles. When fusion occurs between the two 28 Figure 1.12 Schematic representation of lipid mixing assay The stars represent the donor probe (NBD-PE) and the triangles represent the acceptor probe (Rh-PE). Fusion is monitored as an increase of NBD fluorescence. The structures of NBD-PE and Rh-PE are shown (R = PE). o = s = o NBD-PE Rh-PE 29 vesicle systems, the two probes are diluted into the newly formed bilayer. This decreases the resonance energy transfer efficiency, and increases the donor (NBD) fluorescence. By using fluorophore concentrations of less than 1 mol % each of the total lipid concentration, the membrane properties are not significantly altered. In addition, the relative increase of the NBD fluorescence intensity is proportional to the dilution of the probe, allowing relative comparison of membrane fusion (Driessen et al., 1985; Duzgiine§ et al., 1987). 30 1.3 DEOXYRIBONUCLEIC ACID (DNA) 1.3.1 Structural properties of DNA The structural properties of DNA can be classified into three levels of organization. These include the primary, the secondary, and the tertiary structures of DNA. DNA is composed of a sequence of nucleotides joined together via phosphodiester linkages between the 5' hydroxyl group of deoxyribose of one nucleotide and the 3' hydroxyl group of the next nucleotide (Figure 1.13). A nucleotide is a conjugate of a purine or a pyrimidine base, a sugar (deoxyribose for DNA, and ribose for RNA), and a phosphate group. DNA contains four different bases, namely adenine (A), guanine (G), cytosine (C), and thymine (T) which attach to the deoxyribose via a p-glycosidic bond (Figure 1.13). The entire DNA molecule has two strands of nucleotides running anti-parallel in a double helix. The two strands are linked together internally via hydrogen bonding between the purine and pyrimidine bases, with A - T and G - C containing two and three hydrogen bonds respectively. The most common type of secondary structure for DNA in solution is B-DNA, which contains flatly stacked base pairs on top of one another in the inside of the helix, and two grooves (major and minor) on the outside. B-DNA has the characteristic of 10 base pairs per turn with a twist angle of 36° and a length of 3.4 nm. When D N A is subjected to high ionic strength or dehydrated environment, A-DNA is formed. This type of DNA has 11 base pairs per turn with a twist angle of 36°. The base pairs are tilted at an angle of 19° from the normal to the helix axis. Both A and B-DNA are right-handed helices. The third type of DNA, called Z-DNA, contains left-handed helix and has 12 31 Figure 1.13 Structures of deoxyribonucleic acid Bases Purines Pyrimidines Adenine (A) Guanine (G) Thymine (T) Cytosine (C) 32 base pairs per turn with a tilt of 9° from the normal to the helix axis. This type of DNA is common in stretches of DNA composed of mainly poly(G - C) base pairs. The tertiary structures of DNA mainly describe different DNA conformations. DNA can be present in linear or cyclic form. Linear DNA can be formed from cyclic DNA by making a cut in the double stranded helix. In addition, cyclic DNA can form a supercoiled conformation. By breaking only one strand of the supercoiled DNA, an open circular (relaxed circular) DNA is formed. When the cyclic DNA is linked as dimers or trimers via interlocking of the closed DNA rings, this conformation is often called "concatamers" (Figure 1.14). Most naturally occurring DNA is actually circular and has L numbers of helical turns. If this DNA is broken and one of the ends is unwound two times for example, then the numbers of helical turns would be L-2. If this DNA is then re-annealed, the resulting circular D N A will have a region where the two strands are separated in the helix. This underwound molecule has a tendency to form a supercoil conformation with two crossovers in the superhelical turns. Most naturally occurring supercoiled DNA is negatively supercoiled (ie., underwound DNA). Two forms of supercoiled DNA can be generalized. The "plectonemic" form of the supercoiled DNA contains right-handed superhelical turns and has an extended and narrow conformation. This supercoiled DNA often exhibits multiple branches and has a length of about 40 % of the actual DNA (Boles et al., 1990). The plectonemic form often exhibits in solution rather than in a compact state. In contrast, the "solenoidal" form contains tighter, left-handed turns and is often stabilized in a compact 33 34 state. The two forms of supercoiled DNA are interchangeable, and may have different biological functions (Figure 1.14). 1.3.2 Plasmid DNA Plasmid DNA is small, circular, double stranded DNA that is present as extrachromosomal DNA in some bacteria. Different plasmid DNA can be constructed in vitro. Plasmid DNA is often used as a vector for DNA cloning. A typical plasmid often contains a gene expressing antibiotic resistance, a genome with promoter regions critical for the transcription process, and a multiple cloning site where additional DNA can be inserted into the plasmid (Figure 1.15). Plasmid DNA can be produced by transferring the plasmid into bacteria, allowing the bacteria to grow overnight, followed by lysis of the bacteria. The unique properties of plasmids allow them to be easily isolated and purified (Sambrook et al., 1989). Plasmid DNA so obtained is often composed of different conformations depending on the experimental conditions and physical treatment. Plasmid DNA of different conformations can be inter-converted by treatment with different enzymes (Figure 1.14). By treating the crude plasmid DNA with a restriction endonuclease, an enzyme that cuts DNA only at specific base pair sequences, linearized DNA is obtained. If the restriction enzyme only cuts once in the plasmid, a piece of linear DNA containing the same number of base pairs is obtained. When relaxed circular plasmid is treated with DNA gyrase (topoisomerase II), an enzyme that induces negative supercoiling in the DNA molecule, supercoiled plasmid is formed. In contrast, if the supercoiled plasmid is subjected to topoisomerase I, an enzyme that removes negative supercoiling in the DNA molecule, a relaxed circular plasmid is obtained. 35 Figure 1.15 Map of plasmid DNA used in this thesis CMV promoter SV40 poly A CMV promoter SV40 poly A 36 1.4 PROPERTIES OF LIPOSOMES FOR IN VIVO APPLICATION Liposomes designed for systemic in vivo applications must circulate long enough to reach target tissue. The main processes that limit liposome circulation times in vivo are the binding of plasma proteins and lipoproteins, which results in removal by the reticuloendothelial system (RES) (Coleman, 1986; Moghimi and Patel, 1989). The RES is primarily composed of macrophages residing in the liver, spleen, lung, and bone marrow. Plasma protein binding, or "opsonization", of liposomes can also lead to leakage of the interior contents (Kirby et al., 1980; Silverman et al., 1984). It has been demonstrated that the total amount of bound protein on the liposome surface is directly proportional to the liposomal clearance rate (Chonn et al., 1991; Chonn et al., 1992). In order to obtain long circulation times for the injected liposomes, strategies for avoiding the RES have to be employed. This is usually done by modifying the liposomes to reduce opsonization. 1.4.1 Properties of liposomes influencing circulation lifetime Factors that influence the circulation times of liposomes include size, dose, charge and composition. In general, large liposomes (greater than 1 pm diameter) are cleared rapidly compared to smaller systems of less than 200 nm (Juliano and Stamp, 1975). In terms of dosage effect, high liposome injected doses result in longer circulation lifetimes (Poste et al., 1984; Oja et al., 1996). Charged liposomes of either anionic (Chonn et al., 1991; Chonn et al., 1992) or cationic (Senior et al., 1991) nature exhibit faster clearance than 37 the neutral liposomes. Liposomes composed of unsaturated acyl chains have fluid lipid bilayers, which may facilitate opsonization (Kirby et al., 1980; Hunt, 1982). 1.4.2 Poly(ethylene glycol)-conjugated lipids Since protein binding initiates the RES uptake of liposomes, it is reasoned that modification of the surface properties of liposomes to avoid protein binding can lead to prolonged circulation times. One of the common methods to do this is to include a polymer coating on the surface of liposomes. This is usually done by adding small amounts ( 5 - 1 0 mol %) of poly(ethylene glycol) (PEG)-conjugated lipids in the liposome preparation. PEG is a flexible hydrophilic polymer with repeating units of ethylene glycol (-[O-CH2-CH2],,-), which is usually coupled to the head group of a common phospholipid (for example, PE). It has been shown that PEG-PE conjugates can prolong the circulation times of liposomal drug carriers (Blume and Cevc, 1990; Klibanov et al., 1990; Papahadjopoulos et al., 1991; Brannon-Peppas, 1995), and stabilize non-bilayer forming lipids, such as PE, in the lipid mixtures (Holland et al., 1996a). PEG is thought to provide a steric barrier to inhibit protein binding (Lasic, 1994). In addition, it is believed that PEG can reduce cellular uptake directly at the level of the macrophage (Allen et al., 1994). These observations are supported by experiments showing that PEG can sterically inhibit membrane fusion between membrane vesicles (Holland et al., 1996b). 38 1.5 CATIONIC LIPID-MEDIATED DNA TRANSFECTION 1.5.1 Diversity of cationic liposomes Cationic liposomes have been widely used in mediating the delivery of DNA (Feigner et al., 1987; Pinnaduwage et al., 1989; Rose et al., 1991; Feigner et al., 1994; Van der Woude et al., 1995; Liu et al., 1995; Liu et al., 1997; Templeton et al., 1997), RNA (Malone et al., 1989; Weiss et al., 1989), oligonucleotide (Bennett et al., 1992; Colige et al., 1993; Wagner, 1994; Guy-Caffey et al., 1995; Litzinger et al., 1996), and proteins (Debs et al., 1990; Walker et al., 1992) into living cells. These cationic liposomes are usually composed of a mixture of a cationic lipid with a co-lipid, usually DOPE, to enhance transfection activities (Feigner et al., 1994; Mok and Cullis, 1997). Biological cationic lipids are extremely rare; only sphingosine and stearylamine appear in nature. Thus, most of the cationic lipids used for transfection are synthetic. The first two cationic lipids synthesized and used were AyV-dioleyl-Af/V-dimethylammonium bromide (DODAB) (Kunitake and Okahata, 1977) and 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP) (Eibl and Wooley, 1979). It was not until 1987 that Feigner and co-workers reported the first successful cationic lipid-mediated DNA transfection in vitro. Since then, the technology of using cationic lipids in mediating DNA transfer has become widespread. The first cationic liposomes used in mediating efficient DNA transfection were composed of equimolar A/-[l-(2,3-dioleyloxy)propyl]-V,V,A^-trimethylammonium chloride (DOTMA) and DOPE. D O T M A is a quaternary ammonium lipid containing two ether-linked side chains, and has greater chemical stability in aqueous solutions than the comparable ester derivative, DOTAP 39 (Figure 1.16). Since the synthesis of D O T M A requires a multi-step process and is generally of poor yield (Feigner et al., 1987; Feigner et al., 1989), efforts have been made to formulate cationic liposomes using commercially available cationic lipids, such as cetyldimethylethylammonium bromide or chloride (CDAB or CDAC), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), and stearylamine (SA) (Pinnaduwage et al., 1989; Rose et al., 1991; Gustafsson et al., 1995; Tseng et al., 1996). Of these, only formulations containing DDAB can deliver DNA into a variety of cell lines (Rose et al., 1981). Since then, much effort has been devoted towards synthesizing new positively charged lipid molecules, sometimes called "cytofectins" or "cationic amphiphiles", structurally designed to improve transfection potential. In particular, structural modification of existing cationic amphiphiles has yielded a new series of cationic lipids. These include varying the length of the spacer linkage between the ammonium head group and the two hydrophobic side chains (Ito et al., 1990), adding hydroxyalkyl chains on the quaternary amine (such as A^ -(2,3-(dimyristyloxy)propyl]-Af,A^-dimethyl-Af-hydroxyethylammonium bromide, DMRIE) (Feigner et al., 1994), varying the length of the two hydrophobic acyl side chains (Feigner et al., 1994; Solodin et al., 1995), and varying the counter ions of the cationic amphiphiles (Aberle et al., 1996). New cationic lipids have also been developed by synthesizing derivatives from biologically active compounds including the cationic cholesterol derivatives (Leventis and Silvius, 1990), such as 3B-[N-(N',N'-dimethylaminoethane)carbamoyl cholesterol (DC-CHOL) (Gao and Huang, 1991), and the imidazolinium derivatives (Solodin et al., 1995; Liu et al., 1997), which are expected 40 Figure 1.16 Structures of commonly used cationic lipids DOTAP DOTMA DDAB DMRIE DC-CHOL DOSPA DOGS 41 to be biodegradable. These compounds have been shown to have superior transfection properties and less toxicity than conventional synthetic cationic amphiphiles in certain cell lines (Gao and Huang, 1991; Egilmez et al., 1996). Moreover, different polycationic lipids have been synthesized and used as efficient transfection agents. These polycationic lipids are formed from conventional cationic lipids by attaching polycations such as spermine or spermidine (Behr et al., 1989; Remy et al., 1995), or polyelectrolytes such as polylysine (Zhou and Huang, 1994; Gao and Huang, 1996) to the lipid head group. In addition to these permanent positively charged cationic lipids, a series of pH sensitive aminolipids has been designed for future development as transfection reagents (Bailey and Cullis, 1994). Several commercially available transfection reagents have been used for delivering DNA, RNA, oligonucleotides, or proteins to various cell lines (Table 3). Some of the common cationic lipids used in DNA transfection are structurally depicted in Figure 1.16. Another crucial factor that can influence the transfection potency of a cationic lipid system is the choice of co-lipid or "helper" lipid accompanying the cationic lipid. In general, by changing the helper lipid from DOPE to DOPC, a significant reduction in the transfection potency has been reported (Feigner et al., 1994). In contrast, forming the cationic lipid system with equimolar amounts of cholesterol can improve the in vivo transfection potential of the DNA-cationic lipid complexes (Hong et al., 1997; Liu et al., 1997; Templeton et al., 1997). On rare occasions, vesicles formed with pure cationic lipid, most notably DOTAP, can facilitate transfection levels which are similar to or slightly better than their mixtures with DOPE in some cell lines (Feigner et al., 1987; Jarnagin et al., 1992; Stegmann and Legendre, 1997). Interestingly, it has been reported 42 Table 3 Commercially available cationic lipid-based transfection reagents Commercial Name and Manufacturer Lipid Formulation" Charge" Type of Gene Expression Forms of Lipids Molecules transfected Lipofectin (GibcoBRL) DOTMA/DOPE (1:1 w/w) Mono Transient or Stable Small vesicles D N A , R N A , oligonucleotide, proteins LipofectAMINE (GibcoBRL) DOSPA/DOPE (3:1 w/w) Poly Transient or Stable Small vesicles D N A , proteins LipofectACE (GibcoBRL) DDAB/DOPE (1:2.5 w/w) Mono Transient Small vesicles D N A DOTAP (Boehringer Mannheim) DOTAP Mono Transient or Stable Small vesicles D N A , R N A , oligonucleotide, proteins ESCORT (Sigma) DOTAP/DOPE (1:1 w/w) Mono Transient or Stable Small vesicles D N A DMRIE-C (GibcoBRL) DMRIE/CHOL (1:1 MM) Mono Transient or Stable Small vesicles D N A , R N A Cellfectin (GibcoBRL) TMTPS/DOPE (1:1.5 MM) Poly Transient or Stable Small vesicles D N A , R N A , oligonucleotide DOSPER (Boehringer Mannheim) DOSPER Poly Transient or Stable Small vesicles D N A Transfectam (Promega) DOGS Poly Transient or Stable Lipid solution D N A , R N A Tfx-50 (Promega) TD A/DOPE (1:1) Poly Transient or Stable M L V s D N A a (w/w) = weight to weight ratio; (M/M) = molar ratio * Mono = monocationic; Poly = polycationic 43 that alteration of the cationic lipid structure can dramatically alter the co-lipid requirement (Wheeler et al., 1996). Although most of the cationic lipid-based transfection reagents can transfect common cell types (such as COS-7, BHK-21, HeLa, or CHO-K1 cell lines), the extent of gene expression varies for different transfection reagents and cell types. Some reagents are specially designed for particular cell types. For example, DMRIE-C reagent gives the best transfection results for suspension cells such as lymphoid cell lines (Schifferli and Ciccarone, 1996); while the LipofectAMINE reagent can be used to transfect "hard to transfect" cell lines such as human fibroblasts and keratinocytes (Hawley-Nelson et al., 1993). To date, there is no particular type of cationic lipid reagent that can claim to be the universal transfection reagent for all cell types, even though Lipofectin (DOTMA/DOPE), the most common transfection reagent, has been used with many different cell lines. This diverse group of cationic liposomes provides a broad selection for efficient DNA transfection. In the following section, the structures and current models of these DNA-cationic lipid complexes are discussed in detail. 1.5.2 Current models of DNA-cationic lipid complexes DNA-cationic lipid complexes are formed by mixing pre-formed cationic vesicles (transfection reagent) with DNA molecules (such as plasmid DNA containing a gene of interest for delivery). During the past five years, many studies have been carried out to determine the structures of DNA-cationic lipid complexes, as it is believed that understanding these structural features may provide further insight in improving cationic 44 lipid-mediated DNA transfection. Currently, three main models have been proposed for these complexes: (1) the "bead on string" complexes, (2) the "lamellar" complexes, and (3) the "cylindrical" complexes (Figure 1.17). The "bead on string" model was first proposed by Feigner and Ringold (1989). This model consists of several intact cationic liposomes attaching to a linear double stranded DNA as a string (Figures 1.17A). Feigner and Ringold (1989) theoretically estimated that approximately four cationic liposomes are associated with one plasmid DNA molecule. Structures resembling this model were first observed using metal shadowing electron microscopy (EM) at equivalent liposome to DNA ratio (Gershon et al., 1993). However, this observation was not widely taken as a valid representation of the model, since this microscopic technique requires the addition of cytochrome c, which may influence the structures of interest. Moreover, this model fails to consider the effect of plasmid in different conformations, most notably, supercoiled, in which the DNA molecule is condensed in a compact state such that a fraction of the negative charges may not be available for interaction with cationic liposomes. In addition, supercoiled D N A has been reported to have a solution structure in which "branches" of DNA protrude from the corners of a central DNA core, with each branch about equal length to the central core (Figure 1.14) (Boles et al., 1990). Thus, Smith et al. (1993) proposed a revised model, in which this plectonemic form of the supercoiled plasmid DNA acts as a nucleus to allow cationic liposomes to bind, and to form DNA-liposome aggregates consisting of several clustered liposomes. Interestingly, structures resembling this revised model have been observed on a few occasions with different formulations of cationic liposomes. These include DC-CHOL/DOPE (3:2) (Sternberg et al., 1994), DDAB (Bally et al., 45 Figure 1.17 Schematic representation of DNA-cationic lipid complexes Bead on string complexes Cationic liposomes DNA Lamellar complexes iliiliffilffilllliHIl Cationic lipid bilayers iiiiiiiiyyiiiiiiiy iiiiiisifflffiiliil lyyyiyMi IMlIffiMiffillflfl DNA Cylindrical complexes 46 1997), DOTMA/DOPE (1:1) (Mok and Cullis, 1997), and CTAC/DOPE (3:7) (Gustafsson et al., 1995). In the "lamellar model", DNA is trapped between bilayers arranged in a typical M L V pattern. It was first suggested and observed using cryo-TEM (Gustafsson et al., 1995), and later proposed and measured by X-ray diffraction and optical microscopy (Radler et al., 1997; Spector and Schnur, 1997). "Lamellar" entrapped DNA is condensed into a highly ordered phase, which also has been predicted by calculating the balances between the electrostatic repulsion of the DNA molecules with an attractive interaction due to the undulations in the equilibrium packing of the membrane induced by the adsorbed DNA (Dan, 1996; Dan, 1997). It is estimated that the interaxial spacings of this adsorbed DNA are in the range of 2 to 4 nm (Dan, 1996). The observation of heavily fused DNA-lipid assemblies at high DNA content is consistent with the plasmid location between the (fused) membrane bilayers, and thus the entrapped plasmid cannot be easily observed in freeze fracture E M (Sternberg et al., 1994; Hui et al., 1996; Eastman et al., 1997; Mok and Cullis, 1997). The final model involves the DNA strand being surrounded or coated with a monolayer of cationic lipids and/or co-lipids. These cylindrical complexes, sometimes called "spaghetti structures", were first proposed and observed using freeze fracture E M by Sternberg et al. (1994). The presence of hexagonal phase (Hn) forming lipid, such as PE, in the cationic lipid system can provide an additional stabilizing factor for this structural arrangement (May and Ben-Shaul, 1997). Interestingly, these cylindrical complexes are often connected to the DNA-lipid fused aggregates (Sternberg et al., 1994; 47 Hui et al., 1996; Eastman et al., 1997), and have only been observed in systems containing monovalent cationic lipid. The structural features of DNA-cationic lipid complexes are complicated. Different experimental conditions can influence the formation of different structural features in these complexes. These include using different amounts of lipid and DNA, the time of formation, the charge ratio of the system, the type of cationic lipid and co-lipid in the lipid system, the temperature, and the degree of hydration. Nevertheless, understanding the underlying structures of these complexes, and their roles in transfection may enhance the development of future lipid-based gene delivery. 1.5.3 Current hypotheses on mechanisms of lipid-based DNA delivery Another important factor determining efficient lipid-based DNA transfer is the route of entry into the transfecting cells. To date, there are two mechanisms explaining how the DNA entrapped in these complexes is released into cells. The first route is via membrane fusion, while the second route involves endocytosis (Figure 1.18). As mentioned in an earlier section, one of the factors that can induce membrane fusion is charge neutralization. As demonstrated by earlier work, cationic liposomes of different formulations can undergo membrane fusion with anionic model membrane vesicles (Stamatatos et al., 1988; Dtizgune§ et al., 1989; Leventis and Silvius, 1990). The extent of membrane fusion may vary depending on the lipid compositions of both the cationic vesicles and the anionic target membranes. These include the types of cationic lipid and the co-lipid in the cationic vesicles (Leventis and Silvius, 1990), and the anionic lipid content, the acyl chain saturation, and the phospholipid headgroup in the target 48 Figure 1.18 Mechanisms of lipid-based DNA delivery 49 membranes (Bailey and Cullis, 1997). Thus, it is possible that DNA-cationic lipid complexes bearing net positive charge are able to deliver DNA via membrane fusion processes (Figure 1.18A). However recent experimental evidence has suggested the endocytotic pathway is the initial route of entry for the majority of DNA-cationic lipid complexes. The first line of evidence comes from the fact that cell surface binding of cationic liposome alone is insufficient for liposome-cell fusion. This has been shown by interfering with endocytosis either by incubating with chemicals such as ammonium chloride, by incubating in hypertonic media, by depleting the cellular ATP levels, or by lowering the temperature or extracellular pH (Farhood et al., 1995; Wrobel and Collins, 1995; Stegmann and Legendre, 1997). Complementary to these observations, direct evidence of the endocytotic uptake of the complexes has been shown using T E M with either fluorescently labeled lipid and DNA (Zabner et al., 1995; Hui et al., 1996), or with colloidal gold particles complexed to the DNA-lipid particles (Friend et al., 1996). Although the mechanism by which entrapped DNA is released from DNA-cationic lipid complexes remains largely unproven, Xu and Szoka (1996) have proposed that a destabilization process occurs in the endosomal membrane, which leads to the flip-flop of the anionic lipids from the cytoplasmic-facing monolayer. This charge neutralization process eventually leads to membrane fusion and the release of the D N A into the cytoplasm. 50 1.6 DNA ENCAPSULATION Conventional DNA-cationic lipid complexes formed by mixing pre-formed cationic vesicles with plasmid DNA are large, and can be cleared rapidly by the RES in vivo (Huang and Li , 1997). Moreover, the expression of the transfected gene is mainly observed in "first pass" organs such as lung, liver, and spleen (Huang and Li , 1997). That the entrapped DNA is being partially susceptible to DNase and serum degradation (Gershon et al., 1993; Reimer et al., 1995; Hofland et al., 1996), and the toxic side effects of these complexes both in vitro (Harrison et al., 1995) and in vivo (Li and Huang, 1997), suggest the necessity of developing small lipid-based DNA carriers to encapsulate DNA for systemic in vivo applications. 1.6.1 Approaches for encapsulating DNA in liposomes Plasmid DNA has been encapsulated by a variety of methods including reverse phase evaporation (Fraley et al., 1980; Soriano et al., 1983; Nakanishi et al., 1985; Nandi et al., 1986; Alino et al., 1993), Ca 2 + -EDTA chelation (Szelei and Duda, 1989), detergent dialysis in the absence of PEG stabilization (Wang and Huang, 1987), lipid hydration (Lurquin, 1979; Yagi et al., 1994), ether injection (Fraley et al., 1979; Nicolau and Rottem, 1982) and sonication (Jay and Gilbert, 1987; Puyal et al., 1995). Table 4 summarizes the results from these protocols. Each of these methods has some deficiencies in forming ideal plasmid DNA-lipid particles of sufficiently small size (~ 100 nm) and high DNA trapping efficiency. In addition, harsh techniques such as sonication and extrusion after reverse phase or hydration techniques can lead to 51 CD a oo i -H OO „ Os 13 r cu ca .22 o : § O UH 03 "it CL, T3 s i =3. I 2 o I s -1 Z .2 O 13 CB .a < z, .3 Q -o X ) i (N .9 CD a. o . c/1 CJ C U s §• "d- cu < _V2 "3 Q ° *-> T 3 C U B 2; CO 3 - & cu CU T 3 CU OXI in CB i d T3 cu a C U |Li, 'a, 3 W PH PH Q • >>© u o CH w PH W < ro J o X m o u PH w IS 'o cd o 'cu ° O J o ft o O K o P u s w PH o Q e o I-a cu to CO - O C U -t-» ~3 C U S3 c A .2 CU L H cn O »H r v > I (2 5 s « .2 ° f« C3 Q K •i i § .2 § c3 '« .a 5 I * 2 •3 cu CU "ca > C U j i to 52 substantial loss of plasmid DNA and/or DNA breakdown. Thus, novel stabilized plasmid-lipid particles (SPLP) have been developed recently using a detergent dialysis method (Wheeler et al., 1998). 1.6.2 Stabilized plasmid-Iipid particles: construction and characterization Stabilized plasmid-lipid particles (SPLP) are designed to function as in vivo liposomal systems for gene delivery. Earlier work from Reimer et al. (1995) showed that hydrophobic particles can be formed by incubating DNA and cationic lipids in organic solvent. It was reasoned that if this hydrophobic particle was surrounded by an outer monolayer of lipid, the DNA-lipid particle could be stabilized in aqueous medium. Wheeler et al. (1998) first attempted to form these stabilized particles by using detergent dialysis. The detergent was expected to solubilize these hydrophobic DNA-cationic lipid particles, while adding additional neutral phospholipid with subsequent removal of the detergent by dialysis allowed the solubilizing detergent to be exchanged for phospholipid, leaving particles which were stable in aqueous solution. n-Octyl-(3-D-glucopyranoside (OGP), a non-ionic detergent, was chosen because of its high cmc value, which facilitates its subsequent removal by dialysis. Poly(ethylene glycol) conjugated ceramide (PEG-Cer) is included with the phospholipid to prevent aggregation during dialysis. This PEG coating can have the additional effect of reducing serum protein binding in vivo (Harasym et al., 1995), thus providing a basis for prolonged circulation times. In SPLP, PEG-Cer are used instead of the conventional PEG-PE conjugates because the PEG-PE molecule bears a negative charge, which could influence the interaction between the 53 cationic lipid and DNA, as has been noted for other negatively charged lipids (Xu and Szoka, 1996). SPLP have several distinguishing features over other liposomal DNA particles prepared by other techniques (Table 4). First, SPLP are small (~ 70 to 100 nm) as indicated by quasielastic light scattering (QELS) and freeze fracture E M (Wheeler et al., 1998). This property gives SPLP a potential advantage over larger systems, as smaller vesicle systems have extended in vivo circulation times. Secondly, the entrapped plasmid DNA in SPLP is protected by the lipid coating, as shown by the stability of plasmid DNA in serum or following DNase I treatment (Wheeler et al., 1998). Furthermore, SPLP can be purified to yield particles exhibiting a high DNA entrapment efficiency of ~ 60 to 70 %, a high DNA-to-lipid ratio, and which can be concentrated to achieve high plasmid DNA concentrations (1 mg/ml). The final feature of these particles is the ability of the PEG-Cer coating to dissociate, thus increasing transfection potency in a controlled manner. A carrier system must have sufficiently long circulation lifetimes to allow accumulation at disease sites, but must also be able to bind to target cells and, ultimately, destabilize the plasma or endosomal membrane for delivery of the entrapped plasmid. The presence of PEG-lipids stabilizes the hexagonal forming DOPE into the bilayer phase at temperatures above 10 °C (Holland et al., 1996a). Thus, in the absence of PEG-Cer, the SPLP is expected to exhibit a membrane-destabilizing "fusogenic" character. Preliminary in vitro results from Wheeler et al. (1998) indicate that PEG-Cer containing a long acyl anchor chain (C20) have long residence times in SPLP and exhibit lower transfection properties in vitro as compared to PEG-CerCn, which can dissociate rapidly. 54 Thus, by manipulating the PEG-Cer anchor chain lengths in SPLP, "programmable" DNA-lipid particles can be designed for in vivo applications. SPLP have an average of one plasmid per particle residing within an interior aqueous volume (Wheeler et al., 1998). A model for SPLP formation and structure is given in Figure 1.19. SPLP can only be formed at a critical cationic lipid content of approximately 6 - 7 mol %, and a range of PEG-Cer content depending on the PEG-Cer species. For example, at lower cationic lipid contents, no plasmid entrapment is observed, whereas at higher cationic lipid contents, pronounced aggregation is noted. Within the model of Figure 1.19, low cationic lipid content leads to reduced micelle-plasmid association consistent with poor DNA entrapment. In contrast, high cationic lipid contents may allow fusion between the micelle-plasmid aggregates during dialysis. At the critical cationic lipid content however, the positive surface charge density of micelles after association with plasmid is reduced below that needed to associate with other exposed plasmid. Thus instead of having further micelle-plasmid aggregation, only free micelle aggregation occurs. As dialysis proceeds, the plasmid will eventually be coated with lipid. Further dialysis will result in fusion between micelles to eventually produce empty vesicles or in fusion between micelles and the plasmid-lipid particle. Fusion with the particle will result in the deposition of excess bilayer lipid, leading to formation of an associated vesicle in the final SPLP. 55 Figure 1.19 Model of stabilized plasmid-lipid particles formation Model of SPLP formation under various cationic lipid contents proposed in Wheeler et al. (1998). At low cationic lipid content, empty vesicle systems with low plasmid entrapment are obtained; while at high cationic lipid content, plasmid-lipid aggregates are formed (for further details, see text). 56 1.7 THESIS OBJECTIVES The studies of this thesis were first aimed at understanding the structures of conventional DNA-cationic lipid complexes and the relationship to transfection properties, and then describing a novel lipid-based DNA carrier designed for in vivo applications. Chapter 2 describes detailed studies concerning the structural and fusogenic behavior of cationic liposomal systems in the presence of plasmid DNA. The structural and fusogenic properties of different formulations of cationic lipid systems are related to the in vitro transfection potential of the resulting complexes. In Chapters 3 and 4, the preparation of stabilized-plasmid lipid particles (SPLP) are characterized. The influence of the lipid components on both the formation and transfection properties of SPLP is described in Chapter 3, while factors associated with the plasmid components are discussed in Chapter 4. 57 CHAPTER 2 STRUCTURAL AND FUSOGENIC PROPERTIES OF CATIONIC LIPOSOMES IN THE PRESENCE OF PLASMID DNA 2.1 INTRODUCTION Liposomes composed of equimolar mixtures of V-[2,3-(dioleyloxy)propyl]-V,V,./V-trimethylammonium chloride (DOTMA) and l,2-dioleoyl-3-phosphatidylethanolamine (DOPE) can act as agents to mediate intracellular delivery of plasmid DNA into cells. This has been shown to result in efficient transgene expression in vitro (Feigner et al., 1987; Feigner et al., 1989; Lu et al., 1989; Pinnaduwage et al., 1989; Jarnagin et al , 1992), and limited transgene expression in vivo (Nabel et al., 1992; Zhu et al., 1993; Egilmez et al., 1996). In these systems, plasmid DNA is mixed with preformed small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) to form DNA-lipid complexes which interact in turn with target cells (Friend et al., 1996). Considerable effort has been made to characterize the structure of these complexes in order to identify characteristics which correspond to most efficient intracellular delivery of plasmid and subsequent gene expression. Most of these investigations have focused on the morphological features of the complexes as determined by various microscopic techniques, including metal shadowing electron microscopy (EM) (Gershon et al., 1993; Zabner et al., 1995), freeze fracture E M (Sternberg et al., 1994), cryo-transmission E M 58 (Gustafsson et al., 1995), and atomic force microscopy (Wheeler et al., 1996). Little consensus has been reached relating the structures observed to transfection activity. Less attention has been given to the relations between the fusogenic and motional properties of DNA-cationic lipid complexes and transfection potency. Fusion events are clearly integral both to the formation of the complexes themselves as well as subsequent intracellular delivery of DNA. The motional properties of lipids as detected by N M R techniques can give insight into local structure to correlate with fusogenic and transfection behavior. In this work, we examine the structural, motional and fusogenic properties of DOTMA/DOPE LUVs on addition of pCMV5 plasmid, and correlate this information with transfection potential. It is shown that plasmid DNA is a highly potent promoter of fusion between DOTMA/DOPE (1:1) LUVs, forming structures characterized by narrow 3 I P NMR signals characteristic of rapid, isotropic motional averaging. It is suggested that the lipid structures giving rise to this behavior may correspond to non-bilayer lipid arrangements, which facilitate the fusion events associated with transfection by DNA-cationic lipid complexes. 59 2.2 M A T E R I A L S AND M E T H O D S 2.2.1 Lipids and Chemicals 1,2-dioleoyl-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE), l,2-dioleoyl-3-phosphatidylserine (DOPS), 7Y-(7-nitro-2,l,3-benzoxadiazol-4-yl)-l,2-dioleoyl-s«-phosphatidylethanolamine (NBD-PE), and A^lissamine rhodamine B sulfonyl)-l,2-dioleoyl-sn-phosphatidylethanolamine (Rh-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). 3-(7Y,7Y-dimethylamino)-l,2-propanediol, potassium hydride, and iodomethane (methyl iodide) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Magnesium sulfate (MgS0 4), P-mercaptoethanol, potassium acetate, potassium chloride (KCI), sodium chloride (NaCl), anhydrous sodium sulfate (Na 2S0 4), sodium phosphate (Na 2HP0 4), sodium hydroxide (NaOH), glacial acetic acid, and citric acid were obtained from Fisher Scientific (Fair Lawn, NX). Ampicillin, bovine serum albumin (BSA), l-bromo-cz.s-9-octadecene (oleyl bromide), ethylenediaminetetraacetic acid (EDTA), /Y-(2-hydroxyethyl)piperazine-7Y'-2-ethanesulfonic acid (HEPES), lithium chloride (LiCl), lysozyme, poly(ethylene glycol) (PEGgooo), ribonuclease A (RNase), t-octylphenoxypolyethoxy ethanol (Triton X-100), sodium dodecyl sulfate, and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma Chemical Co. (St. Louis, MO). 14C-labeled cholesteryl hexadecyl ether ( 1 4 C-CHE) was purchased from Dupont N E N Products (Boston, MA). Chlorophenol red galactopyranoside (CPRG) was purchased from Boehringer Mannheim (Germany). Deuterium oxide (D 20) was obtained from MSD Isotopes (Montreal, Ontario). Bacto-tryptone and bacto-yeast extract were purchased from Difco Laboratories (Detroit, MI). All organic solvents were purchased 60 from Fisher Scientific (Nepeau, Ontario). Freon 22 was obtained from Allied Chemical Ltd. (Mississauga, Ontario). Silica gel 60 (particle size 63-200 urn, 70-230 mesh) was purchased from VWR Scientific (Edmonton, Alberta). Agarose was purchased from Bio-Rad (Richmond, CA). Glycerol and glucose were obtained from B D H (Toronto, Ontario). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, EcoRI, and 1 kilobase DNA ladder were purchased from GibcoBRL (Burlington, Ontario). Baby hamster kidney (BHK) cells (BHK 21) were obtained from American Tissue Culture Collection (ATCC CCL-10, Manassas, VA). Scintillation fluid (Ultima Gold grade) was obtained from Packard Instrument Co. (Downers Grove, IL). Distilled water was purified by Corning Mega-Pure MP-4S system. 2.2.2 Synthesis of A^[2,3-(dioleyloxy)propyl]-AVV7Ar-trimethylammonium (DOTMA) D O T M A was synthesized as described previously (Feigner et al., 1987) with the following modified procedure. A mixture of 3-(dimethylamino)-l,2-propanediol (0.92 g, 1.49 mmol), potassium hydride (0.36 g, 3.14 mmol), and oleyl bromide (1.50 g, 4.48 mmol) in xylenes (20 mL) was stirred at room temperature and reduced pressure (30 mm Hg) for 30 min. After heating the mixture to 50 °C with a condenser and continuous stirring for an additional 15 min, the mixture was heated to reflux under continuous nitrogen flow at atmospheric pressure for 4 hours. After cooling and adding hexane (150 mL), distilled water (150 mL) was added dropwise to the mixture. The reaction mixture was then extracted until neutrality was reached. After drying the organic layer with anhydrous sodium sulfate, the crude intermediate product was concentrated under rotary 61 evaporation and was purified using a silica gel column, with stepwise elution of 2:1 diethyl ether/hexane and then 4:2:1 diethyl ether/hexane/ethanol. The intermediate product, 2,3-dioleyloxy-l-(dimethylamino)propane, was obtained as a colorless oil (0.55 g, 60 % yield; T L C , Rf = 0.40 with 4:2:1 diethyl ether/hexane/ethanol), and the structure was confirmed by 200 MHz *H NMR. The final product was prepared by stirring the intermediate product (0.42 g, 0.67 mmol) with methyl iodide (0.68 g, 4.79 mmol) in dichloromethane (40 mL) for 20 hours in the dark. After rotary evaporation, the residue was dissolved in 60 mL dichloromethane and was repeatedly extracted with aliquots of 40 mL 1.0 M NaCl until the iodide form of D O T M A (TLC, R f = 0.35 with 6:1 dichloromethane/methanol) was converted into the chloride form of D O T M A (TLC, Rf = 0.22 with 6:1 dichloromethane/methanol). Column chromatography on silica gel, eluting with 1:1 hexane/ethanol and then 6:1 dichloromethane/methanol, gave 0.30 g (67 % yield) of pure product. The structure of D O T M A was confirmed by 200 MHz *H N M R and low resolution power liquid secondary ion mass spectroscopy (Kratos Concept II H) with parent ion mass of 635 atomic mass unit. 2.2.3 Plasmid preparation Plasmid DNA (pCMV5) (Andersson et al., 1989) was grown in Escherichia coli (DH5a) and was selected by the resistance to ampicillin. pCMV5 was isolated by alkali lysis and purified by PEG precipitation (Sambrook et al., 1989). Briefly, 3.0 L of saturated bacterial cultures were collected by centrifugation and resuspended in 108 mL of 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA. After the addition of a tip in spatula of solid lysozyme, and then 240 mL of freshly prepared 0.20 M NaOH/1 % sodium dodecyl 62 sulfate with gentle mixing for 15 min, 180 mL of ice-cold 5.0 M potassium acetate/glacial acetic acid/distilled water (volume ratio of 60:11.5:28.5) was distributed evenly to the lysate, which was then cooled in ice for 15 min, centrifuged at 5,000 rpm for 30 min, and filtered through a layer of cheesecloth. An equal volume of isopropanol was added to the supernatant for precipitation. After centrifugation, the pellet was rinsed with 70 % ethanol and resuspended in 18 mL T E (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). An equal volume of ice-cold 5.0 M LiCl was then added for precipitation; and an equal volume of isopropanol was added to the resulting supernatant for re-precipitation. The resulting pellet was washed with 70 % ethanol and was resuspended in 3.0 mL T E with 100 pL DNase-free pancreatic RNase (10 mg/mL) for 2 hours at room temperature. 3.0 mL of 1.6 M NaCl with 13 % (w/v) PEGgooo was added and the plasmid was recovered by centrifugation at 11,000 g for 5 min. The pellet was extracted once with phenol, once with phenol/chloroform, and once with chloroform. The aqueous layer was treated with 600 pL 3.0 M sodium acetate and two volumes of ethanol. The precipitated plasmid was washed with 70 % ethanol and was resuspended in distilled water. The purity of pCMV5 was confirmed by 1 % agarose gel electrophoresis with restriction endonuclease EcoR I digest. Since one nucleotide contains only one phosphate group, the concentration of pCMV5 was determined by using standard phosphorus assays (Fiske and Subbarow, 1925) and was expressed as phosphorus concentration of pCMV5. A similar procedure was used for the preparation of pCMVPgal. 63 2.2.4 Preparation of large unilamellar vesicles (LUVs) Mixture of lipids dispensed in chloroform were dried under a stream of nitrogen gas with continuous vortex-mixing. The residual solvent was removed under high vacuum for 2 hours. The resulting lipid films were hydrated with appropriate buffers and then freeze-thawed five times to produce homogenous multilamellar vesicles system (MLVs). Large unilamellar vesicles system (LUVs) were obtained by extruding MLVs 10 times through two 100 nm pore size polycarbonate filters (Costar Nuclepore polycarbonate membrane) under a nitrogen pressure of 300 - 400 psi (Mayer et al., 1986). The size of LUVs was checked with a Nicomp Model 270 submicron particle sizer using quasielastic light scattering techniques. Phosphorus assays were used to quantify phospholipid concentrations (Fiske and Subbarow, 1925). 2.2.5 Lipid-Mixing Fusion Assay Fusion was determined by the decrease in resonance energy transfer (RET) resulting from dilution of fluorescent probe (Struck et al., 1981). Exchange of labeled lipids between two populations of vesicles, even in aggregated systems has been reported to be negligible (Hoekstra, 1982; Diigiines et al., 1987). LUVs of desired composition were prepared in 20 mM HEPES (pH 7.4) and were diluted to 10 mM total lipid. Fluorescently labeled LUVs were prepared using DOTMA/DOPE (1:1) or DOTMA/DOPC (1:1) and also containing 0.5 mol % each of NBD-PE and Rh-PE. To study the effects of NaCl, citrate and pCMV5 on fusion of DOTMA/DOPE (1:1) and DOTMA/DOPC (1:1) LUVs, a mixture of 1:9 labeled to non-labeled LUVs was prepared. Then, 100 uL of the mixed LUVs was added to 1850 uL of 20 mM HEPES (pH 7.4) in a cuvette with continuous 64 stirring, and the fluorescence intensity (F) was monitored over time. Fusion was induced by the addition of an aliquot of appropriate anions (50 pL) at 30 s after putting the mixed vesicles in the cuvette. Excitation and emission wavelengths were 445 nm and 535 nm, respectively, and a 530 nm emission cutoff filter was used. A blank assay of adding 50 pL of 20 mM HEPES (pH 7.4) was used as reference for zero fluorescence (F0). Maximum fluorescence intensity (F m a x ) was measured by the addition of 4 % Triton X -100 (40 p.L). A mock complete mixing system of LUVs containing 0.05 mol % each of fluorescent probes was also prepared. The fluorescence intensity before (Fb) and after (Fa) the addition of 40 pL 4 % Triton X-100 in this mock sample was taken into consideration when calculating the percent change in fluorescence (% AF/AF m a x ) for each point in the fluorescence time course. % A F / A F m a x = {[(F - F 0 ) / (F m a x - Fo)]100}(Fb/Fa) [1] For studying fusion of DOTMA/DOPE or DOTMA/DOPC LUVs with DOPS systems, 10 pL of fluorescently labeled DOTMA/DOPE (1:1) or DOTMA/DOPC (1:1) LUVs (10 mM total lipid) was added to 1940 pL of 20 mM HEPES (pH 7.4) in a cuvette with continuous stirring. At 30 s, 50 pL of appropriate composition of non-labeled DOPS/DOPE or DOPS/DOPC LUVs (10 mM total lipid) was added to induce fusion. Mock samples for different composition containing 0.083 mol % each of fluorescent probes were prepared. An assay of 50 pL of 20 mM HEPES (pH 7.4) was used for zero fluorescence (Fn u) while fluorescence of the appropriate mock samples was taken to be the maximum fluorescence (Fm a x). The percent change in fluorescence was then calculated as % AF/AFmax = [(F - F n u ) / (F m a x - Fnu)]100 [2] 65 for each point in the fluorescence time course. For studying the interaction between the DNA-lipid complexes and anionic model membrane system, 10 uL of fluorescently labeled DOTMA/DOPE (1:1) or DOTMA/DOPC (1:1) LUVs (10 mM total lipid) was added to 1890 uL of 20 mM HEPES (pH 7.4) in a cuvette with continuous stirring. An aliquot of 50 uL of serial diluted pCMV5 stocks was added at 30 s in each assay. At 90 s, 50 uL of 10 mM non-labeled DOPS/DOPE (1:1) or DOPS/DOPC (1:1) LUVs was added. The data processed from this experiment was right-shifted for 60 s (i.e. time zero was taken as at 60 s time point). The fluorescence intensity obtained after the addition of pCMV5 to the labeled LUVs was taken as reference for each assay (Fn u). Maximum fluorescence intensity (F m a x ) was measured by the addition of 25 uL 4 % Triton X-100. A mock sample of LUVs containing 0.083 mol % each of fluorescent probes was used, and the % A F / A F m a x was calculated as % A F / A F m a x = {[(F - F n u ) / (F m a x - Fnu)]100}(Fb/Fa) [3] for each point in the fluorescence time course. All the fluorescence measurement were monitored using a Perkin Elmer Luminescence spectrometer LS50 which is thermostated at 25 °C. 2.2.6 3 1 P NMR Spectroscopy Solid state broad-band decoupling P NMR spectra were recorded at 81.02 MHz on a Bruker MSL 200 spectrometer, using a 4.0 L I S pulse and a 1.5 s repeat time. The free induction decay (FID) was accumulated over 1500 - 2000 scans and was Fourier transformed with 50 Hz line-broadening. For studies showing the effect of D O T M A on DOPE, various compositions of freeze-thawed DOTMA/DOPE (50 umol, 33 mM 66 DOPE) MLVs were hydrated in 20 mM HEPES/D 2 0 (pH 7.4). Temperature was controlled by a Bruker Variable Temperature 1000 Unit at settings with increments of 10 °C. Temperature under 40 °C was controlled with a liquid nitrogen flow system; while temperature above 40 °C was maintained with a nitrogen gas flow system. For the experiments showing the effects of different anions on DOTMA/DOPE or DOTMA/DOPC systems, LUVs of DOTMA/DOPE (1:1) or DOTMA/DOPC (1:1) (50 pmol each, 100 mM total lipid) in 20 mM HEPES/D 2 0 (pH 7.4) were prepared as described earlier. Then, sequential addition of the appropriate amounts of different anions to each sample was used to induce changes in lipid organization. Temperature was maintained at 25 °C using a liquid nitrogen flow system. The FID of the resuspended pellet and supernatant of DNA-cationic lipid complexes were accumulated over 10,000 -20,000 scans. For the separation of DNA-cationic lipid complexes, an appropriate amount of plasmid DNA was added to a population of LUVs prepared in 20 mM HEPES. The mixture was centrifuged at 11,000 g in Sorvall RC-5B superspeed centrifuge for 15 min. The pellet was washed twice with distilled water and centrifuged before resuspending in 20 mM HEPES/D 2 0. All 3 1 P NMR spectra were locked with D 2 0 . A mixture of phosphoric acid/D20 was used as reference for chemical shifts in all 3 1 P N M R spectra. 2.2.7 Freeze Fracture Electron Microscopy Samples of DOTMA/DOPE (1:1) and DOTMA/DOPC (1:1) (30 mM total lipid) LUVs in 20 mM HEPES (pH 7.4) were prepared as described earlier. 50 pL aliquots of lipid sample were titrated with the appropriate amount of NaCl, citrate, or pCMV5. After incubating at room temperature for 15 min, 33.5 pL of glycerol was added as 67 cryoprotectant to make a final glycerol concentration of 25 % by volume in each sample. The final concentration of total lipid in each sample was 11 mM (0.75 umol each of D O T M A and DOPE or DOPC). After physical mixing and incubating for another 15 min, a 2.0 u.L droplet of the sample was pipetted onto a flat-top gold support disc which was then plunged into liquid nitrogen-cooled liquid freon 22 (monochlorodifluoromethane; freezing point -160 °C). After 5 seconds, the sample was transferred onto a specimen table immersed in liquid nitrogen prior to insertion into the freeze fracture apparatus (Balzers; BAF400D). Fracturing was performed at -110 °C with a vacuum of 10"6to 10"7 torr. Immediately after the fracturing, 2 nm coating of platinum-carbon at an angle of 45° and then a 20 nm coating of carbon at an angle of 90° were applied. The gold support disc was then warmed to room temperature and was submerged into distilled water for replicas removal. Replicas were cleaned overnight in commercial bleach solution and were rinsed two times with distilled water before mounting onto a grid using a platinum transfer loop. The dried replicas were examined under a transmission electron microscope (Jeol; JEM-1200EX). 2.2.8 Separation and quantification of DNA-cationic lipid complexes DNA-cationic lipid complexes were formed by incubating appropriate amounts of preformed cationic vesicles with 20 L i g pCMVPgal to obtain the desired charge ratio (+/-) in 250 uL distilled water. Trace amounts of 3H-pCMVPgal and 1 4 C - C H E were used as markers for this experiment. The resulting mixture was incubated at room temperature for 15 min and was then centrifuged at 11,000 g in Sorvall MC-12V microcentrifuge for 15 min. The supernatant was pipetted out for storage, while the pellet was resuspended in 68 250 LIL distilled water. Mock samples for each charge ratio without centrifugation were also prepared. Aliquots of 50 pL from samples of the pellet and the supernatant were counted for radioactivity, and were compared with that from mock samples for the percentage of 3 H DNA and 1 4 C lipid recovery, respectively. 2.2.9 In vitro DNA transfection on B H K cells All DNA transfection procedures were carried out in a laminar flow hood (Forma Scientific). DNA-cationic lipid complexes were prepared as described above except 0.5 pg of pCMVPgal was used for each transfection preparation. Appropriate amounts of vesicles were used for each charge ratio. Transfections were performed in triplicate. Standards of P-galactosidase were prepared by two-fold serial dilutions of 200 milliunits P-galactosidase with 0.5 % bovine serum albumin (BSA) in phosphate buffered saline (PBS) (pH 8.0). A unit of P-galactosidase will hydrolyze 1.0 pmol of onitrophenyl-p-D-galactoside to o-nitrophenol and D-galactose per minute at pH 7.3 and 37 °C. A similar transfection protocol has been published elsewhere (Feigner et al., 1994). First, baby hamster kidney (BHK) cells (BHK 21) cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10 % fetal bovine serum (FBS) and 100 units of penicillin and 100 pg streptomycin as antibiotics were plated onto a 96 well microtiter plate, at a cell density of 2 x 104 cells per well. The plate was then incubated for 20 hours at 37 °C with 5 % CO2. For each well, appropriate amounts of DNA-lipid complexes (preformed for 30 min) were diluted with DMEM/FBS. Aliquots of 100 pL of DNA-lipid complexes were plated and the resulting plate was incubated at 37 °C, 5 % CO2 for 4 hours. After the removal of the transfected media, 100 pL of DMEM/FBS was added and cells were further 69 incubated for 20 hours at 37 °C, 5 % CO2. Cells were lysed by adding 50 u.L of lysis buffer containing 0.1 % Triton X-100 in 250 mM phosphate buffer (pH 8.0) and were left at -70 °C for about 30 min to ensure complete lysis. After thawing, the samples were subjected to P-galactosidase assay. Briefly, 50 uL of PBS containing 0.5 % BSA or 50 uL of appropriate P-galactosidase standard was added. Color development was induced by adding 150 uL substrate buffer containing chlorophenol red galactopyranoside (CPRG) (1 mg/mL), 60 mM Na 2 HP0 4 , 1 M MgS0 4 , 10 mM KC1, and 50 mM p-mercaptoefhanol, and was measured at 540 nm using a Microplate Autoreader EL-309 (Bio-Tek Instruments). For the transfection of samples from the resuspended pellet and the supernatant of DNA-lipid complexes after centrifugation at 11,000 g, trace amounts of 3H-pCMVpgal and 1 4 C - C H E were included for quantification. 70 2.3 RESULTS 2.3.1 DOTMA can stabilize DOPE into a bilayer organization The effect of D O T M A on the structural behavior of DOPE in aqueous dispersions of mixtures of D O T M A and DOPE was investigated employing 3 1 P N M R techniques. DOTMA/DOPE dispersions with increasing D O T M A content (from 0 % to 80 %) were prepared in 20 mM HEPES buffer (pH 7.4). For each sample, 3 1 P N M R spectra were recorded from 10 °C to 70 °C in increments of 10 °C. As shown in Figure 2.1, at both 20 °C and 40 °C, aqueous dispersions of pure DOPE exhibit a typical hexagonal (Hn) phase 3 1 P N M R signal (Cullis and de Kruijff, 1979), which is characterized by a high field shoulder and a low field peak where the separation between the shoulder and peak is about half of that observed for the bilayer 3 1 P NMR signal. As the D O T M A content increases, an increased bilayer 3 1 P N M R component, characterized by a high field peak and a low field shoulder, is observed. DOPE was partially stabilized into a bilayer organization at temperatures up to 20 °C in the presence of 20 % DOTMA; whereas at 50 % D O T M A content, DOPE was stabilized into a bilayer organization up to 60 °C. With an 80 % D O T M A content, bilayer organization was observed over the entire temperature range studied. Thus for a DOTMA/DOPE (1:1) system, which is the composition of the commercially available Lipofectin reagent, DOPE is stabilized into a bilayer organization both at room and physiological temperatures. The ability of D O T M A to stabilize DOPE into a bilayer organization is similar to the ability of lipids such as phosphatidylcholine (PC) as well as anionic phospholipids, such as phosphatidylserine (PS) to stabilize PE into a bilayer organization (Cullis and de Kruijff, 1979). 71 Figure 2.1 Influence of D O T M A content in D O T M A / D O P E M L V s 3 1 P N M R spectra of freeze-thawed DOTMA/DOPE MLVs in 20 mM HEPES (pH 7.4) with increasing D O T M A content (0 %, 20 %, 50 %, and 80 %) at (A) 20 °C, and (B) 40 °C. Sample preparation and NMR parameters are described in Materials and Methods. i i,. ••• i • 50 0 -50 50 0 -5< PPM PPM 72 2.3.2 pCMV5 can trigger membrane fusion between D O T M A / D O P E LUVs The ability of equimolar amounts of D O T M A to stabilize DOPE in a bilayer organization allows stable large unilamellar vesicles to be made. Addition of plasmid DNA results in formation of the DNA-lipid complexes used for transfection. It is of interest to examine the fusogenic behavior of DOTMA/DOPE (1:1) LUVs in response to the presence of plasmid DNA. This was performed employing the lipid mixing assay detailed in section 2.2.5. DOTMA/DOPE (1:1) LUVs containing 0.5 mol % each of NBD-PE and Rh-PE and non-labeled 1:1 DOTMA/DOPE vesicles were prepared. When the labeled vesicles fuse with non-labeled vesicles, dilution of the fluorescent probes results in an increase in NBD-PE fluorescence. Fusion between DOTMA/DOPE LUVs on addition of pCMV5 plasmid is shown in Figure 2.2A. The number beside the fluorescence profile is the cation to anion charge ratio. For the titration of pCMV5, the charge ratio is determined as the moles of D O T M A to that of phosphorus in the plasmid. As the pCMV5 content increases, increased fusion is observed. A comparison between plasmid induced fusion and that induced by other anions is provided by Figures 2.2B and C. For the titration of citrate and NaCl, the number beside each fluorescence profile is the respective anion concentration. To achieve 80 % fusion at 3.5 min after addition of anions, 0.063 mM phosphorus concentration of pCMV5 (charge ratio = 3.0) is required; whereas 0.93 mM citrate carboxyl (charge ratio = 0.20) and 800 mM NaCl (charge ratio = 2.3 x 10"4) is required, respectively. Thus, on a per molecule basis, pCMV5 is about 5.0 x 104 fold more effective at inducing membrane fusion between 1:1 DOTMA/DOPE LUVs than citrate, which in turn is about 2.7 x 103 fold more effective than NaCl. 73 Figure 2.2 Fusogenic behavior of DOTMA/DOPE vesicles in the presence of anions Effect of (A) pCMV5 plasmid, (B) citrate, and (C) NaCl on fusion of DOTMA/DOPE (1:1) vesicles in 20 mM HEPES (pH 7.4) at 25 °C. Vesicles were prepared with and without 0.5 mol % each of NBD-PE and Rh-PE. The labeled and unlabeled vesicles were mixed in a 1:9 ratio and diluted to 0.38 mM total lipid. Different anions were added at 30 s. The concentration of citrate and NaCl (mM), and the charge ratio of DNA-cationic lipid complexes are indicated, respectively. System without DNA is indicated by "n.a.". The charge ratio is defined as the moles of D O T M A to moles of phosphorus in pCMV5. 120 8 100 3 80 60 40 3 c I 20 u 0 -20 3.0 5.0 7.5 15 n.a. 30 60 90 120 150 180 210 240 Time (s) §100 Time (s) 120 S 100 -20 J , _ J 0 30 60 90 120 150 180 210 240 Time (s) 74 2.3.3 Addition of pCMV5 to DOTMA/DOPE (1:1) LUVs causes formation of large lipid structures characterized by isotropic motional averaging The structural characteristics of the DNA-cationic lipid complexes are of obvious interest. 3 1 P N M R was used to identify the behavior of DOPE in the complexes; and freeze fracture electron microscopy was used to examine local structure. As shown in Figure 2.3, DOTMA/DOPE (1:1) LUVs at 25 °C exhibit a narrow "isotropic" 3 1 P N M R signal with peak width at half height of about 7.0 ppm. Addition of pCMV5 to a charge ratio of 1.0 does not cause significant broadening (Figure 2.3 A). An additional broad 3 1 P N M R signal, with a half width of approximately 100 ppm was detected underneath this broad isotropic signal (data not shown). Two lines of evidence indicate that this broad 31 line originated primarily from plasmid phosphorus. First, the intensity of the narrow P N M R component was not significantly affected by the addition of plasmid DNA. At the 1.0 charge ratio, for example, the intensity of the narrow component was approximately 92 % of that observed prior to addition of plasmid. Second, the addition of pCMV5 plasmid to pure D O T M A LUVs (charge ratio 1.0) resulted in a broad spectral feature with a width of approximately 100 ppm (data not shown). Thus, the observed isotropic 3 1 P NMR signal and particle sizes of microns or larger from quasielastic light scattering (QELS) measurement indicate large systems with rapid isotropic motion of the phospholipid. This can correspond to a variety of lipid structures, including small bilayer vesicles or non-bilayer structures such as cubic phases (Cullis et al., 1985). In order to visualize the structures formed on addition of plasmid to DOTMA/DOPE (1:1) LUVs, freeze fracture E M studies were performed. In the absence 75 Figure 2.3 3 1 P N M R of D O T M A / D O P E LUVs in the presence of anions 3 1 P NMR spectra of DOTMA/DOPE (1:1) LUVs with sequential addition of pCMV5 plasmid (A), citrate (B), and NaCl (C) in HEPES (10-20 mM; pH 7.4) at 25 °C. The charge ratio of DNA-cationic lipid complexes and the concentration of citrate and NaCl (mM) are indicated, respectively. System without pCMV5 is indicated by "n.a.". The charge ratio is defined as the moles of D O T M A to moles of phosphorus in pCMV5. 76 of plasmid, vesicles with an average size of 100 nm were observed (Figure 2.4A). When pCMV5 plasmid was added to achieve a +/- charge ratio of 600, the majority of the vesicles remained in dispersion (data not shown). At a charge ratio of 20, most vesicles aggregated into small clusters with little evidence of fusion (Figure 2.4B). This is consistent with the 20 % lipid mixing observed employing the fusion assay (Figure 2.2A). At a charge ratio of 4.0, similar aggregated vesicles fuse into complexes with sizes ranging from 200 to 1000 nm (Figure 2.4C). Structures that resemble lipidic particles (Verkleij et al., 1979) arranged in a row were occasionally observed (arrow 1). The observation of large complexes correlates with the results of the fluorescent fusion assay, in which 80 % fusion was observed at a charge ratio of 3.0 (Figure 2.2A). At charge ratios of 1.0 and lower, similar aggregates of fused lipid assemblies were observed (data not shown). The structural behavior of DOTMA/DOPE LUVs in the presence of other anions was also examined using 3 1 P NMR. Titration of DOTMA/DOPE LUVs with chloride and citrate were performed at pH 7.4, and the respective 3 1 P NMR spectra are shown in Figures 2.3B and C. At a high NaCl concentration of 300 mM, a mixture of bilayer, isotropic, and Hn signal was observed (Figure 2.3C). The size of the system increased from about 100 nm to about 600 nm as detected employing QELS. Further addition of NaCl (0.7 to 2.0 M) induced formation of the Hn phase. In contrast, mixtures of bilayer and Hn signals were observed in the range of 10 to 50 mM citrate (Figure 2.3B). Further addition of citrate induced complete Hn structural organization in the DOTMA/DOPE LUVs. 77 Figure 2.4 Freeze fracture E M of DOTMA/DOPE LUVs in the presence of plasmid DNA Freeze fracture electron micrographs of D O T M A / D O P E (1:1) L U V s in HEPES buffer (10 - 20 m M , pH 7.4) with different amount of p C M V 5 . D O T M A / D O P E (1:1) L U V s (A) in the absence of p C M V 5 ; (B) in the presence of p C M V 5 (charge ratio of 20); and (C) in the presence of p C M V 5 (charge ratio of 4.0) are shown. The bar on all electron micrographs represents 200 nm and the shadow direction is running from bottom to top. The original magnification was 20,000 X . 78 2.3.4 Addition of pCMV5 to DOTMA/DOPC (1:1) LUVs causes formation of large bilayer lipid structures Previous studies have shown that the DOPE "helper" lipid is essential for efficient transfection employing DNA-cationic lipid complexes (Leventis and Silvius, 1990; Farhood et al., 1995). This likely arises from the well known preference of DOPE for non-bilayer structures, some of which may play a direct role in membrane fusion (Cullis et al., 1985; Litzinger and Huang, 1992). It is therefore of interest to examine the structural and fusogenic properties of DNA-cationic lipid complexes in which DOPE is replaced by the bilayer forming lipid, DOPC. As shown in Figure 2.5A, considerable fusion was observed when pCMV5 plasmid was added to DOTMA/DOPC (1:1) LUVs. Compared with DOTMA/DOPE (1:1) LUVs, DOTMA/DOPC (1:1) LUVs require about 1.5 to 2.0 fold more pCMV5 to obtain the same level of lipid mixing. In 3 1 P N M R studies, when pCMV5 was added to DOTMA/DOPC (1:1) LUVs, an isotropic signal was maintained at charge ratios > 8.3; however at higher pCMV5 content (lower +/- charge ratios), mixtures of isotropic and bilayer signals were detected (Figure 2.6A). Again quasielastic light scattering studies revealed a size increase to microns or larger at high pCMV5 levels (charge ratio of 1.0). Freeze fracture E M studies on these complexes revealed similar behavior as for the DOPE containing systems; however in the large fused systems obtained at low charge ratios, structures which could correspond to lipidic particles were not observed (Figure 2.7). The effects of citrate and chloride on the structural and fusogenic behavior of DOTMA/DOPC (1:1) LUVs were also examined. As shown in Figure 2.5B, about 103 times as much citrate in DOTMA/DOPC LUVs is required to induce the same level of 79 Figure 2.5 Fusogenic behavior of D O T M A / D O P C vesicles in the presence of anions Effect of (A) pCMV5 plasmid, (B) citrate, and (C) NaCl on fusion of DOTMA/DOPC (1:1) vesicles in 20 mM HEPES (pH 7.4) at 25 °C. Vesicles were prepared with and without 0.5 mol % each of NBD-PE and Rh-PE. The labeled and unlabeled vesicles were mixed in a 1:9 ratio and diluted to 0.50 mM total lipid. Different anions were added at 30 s. The concentration of citrate and NaCl (mM), and the charge ratio of DNA-cationic lipid complexes are indicated, respectively. System without pCMV5 is indicated by "n.a.". The charge ratio is defined as the moles of D O T M A to moles of phosphorus in pCMV5. 2000 0 30 60 90 120 150 180 210 240 Time ( s ) 80 Figure 2.6 3 1P NMR of DOTMA/DOPC LUVs in the presence of anions 3 1 P NMR spectra of DOTMA/DOPC (1:1) LUVs with sequential addition of pCMV5 plasmid (A), citrate (B), and NaCl (C) in HEPES (10-20 mM; pH 7.4) at 25 °C. The charge ratio of DNA-cationic lipid complexes and the concentration of citrate and NaCl (mM) are indicated respectively. System without pCMV5 is indicated by "n.a.". The charge ratio is defined as the moles of D O T M A to moles of phosphorus in pCMV5. 81 Figure 2.7 Freeze fracture E M of D O T M A / D O P C LUVs in the presence of plasmid DNA Freeze fracture electron micrographs of DOTMA/DOPC (1:1) LUVs in HEPES buffer (20 mM, pH 7.4) in the presence of pCMV5 at charge ratio of 4.0. The bar on the electron micrograph represents 200 nm and the shadow direction is running from bottom to top. The original magnification was 20,000 X. 82 lipid mixing as for DOTMA/DOPE LUVs. No significant lipid mixing was observed at NaCl concentrations of up to 2.0 M (Figure 2.5C), in agreement with the observation of an isotropic 3 1 P NMR signal (Figure 2.6C) with no significant increase in the size of vesicles as detected by QELS and freeze fracture techniques (data not shown). However, when citrate was added, unusual behavior was observed. A mixture of bilayer, isotropic, and H n 3 1 P N M R signal was detected at 10 to 100 mM citrate (Figure 2.6B), and at 150 mM citrate or higher, an isotropic lineshape was observed. The size of the DOTMA/DOPC (1:1) system in the presence of > 150 mM citrate is at least 100 fold larger than that of the untreated vesicles as detected by QELS, again suggesting the formation of cubic or other non-bilayer structures giving rise to isotropic NMR spectra. 2.3.5 D O T M A / D O P E LUVs fuse with anionic vesicles and pCMV5 inhibits such fusion In order for transfection to occur, the DNA-cationic lipid complexes must first fuse with the target cell membrane, which bears a negative charge due to sialic acid residues in the glycocalyx. As a simple model for this interaction, fusion between DOTMA/DOPE (1:1) LUVs or DNA-cationic lipid complexes, and anionic LUVs containing phosphatidylserine (PS) was examined. As shown in Figure 2.8, no fusion was observed when DOTMA/DOPE LUVs were incubated with DOPC LUVs. As the DOPS content was increased to 2 mol % in the DOPS/DOPC LUVs, the amount of fusion with DOTMA/DOPE LUVs increased considerably. When pure DOPS LUVs were used, 65 % fusion at 3.5 min after addition was observed. If DOPE was substituted for DOPC in the 83 Figure 2.8 Fusogenic behavior of cationic vesicles with anionic model membrane vesicles Fusion of DOTMA/DOPE (1:1) LUVs with various composition of DOPS/DOPC LUVs in 20 mM HEPES (pH 7.4) at 25 °C. The percentage of DOPS content in each vesicles system is indicated. Fusion assays were carried out as described in Materials and Methods. 84 DOPS containing vesicles, the rate and extent of fusion increased markedly (data not shown). The influence of pCMV5 on fusion between cationic and anionic LUVs is illustrated in Figure 2.9. In these experiments, the fluorescently labeled DOTMA/DOPE LUVs were first mixed with pCMV5, and the complexes were then incubated with non-labeled DOPS/DOPC (1:1) vesicles. When no pCMV5 plasmid was present, 60 % fusion was observed; however at higher pCMV5 levels corresponding to charge ratios of 0.91 or smaller, fusion was markedly inhibited. Fusion of the same complexes with DOPS/DOPE (1:1) LUVs showed similar behavior. In the absence of plasmid, 80 % fusion was observed; whereas the presence of pCMV5 at a charge ratio of 1.0 reduced the fusion index to 60 %, and only 5 % fusion was observed at a charge ratio of 0.83 (data not shown). 2.3.6 Complexes formed by addition of plasmid DNA to cationic LUVs can be separated into two fractions by centrifugation In an attempt to understand the origin of the isotropic 3 1 P NMR resonance observed for pCMV5-DOTMA/DOPE (1:1) at a charge ratio of 1.0 (Figure 2.3), the complexes were separated into two fractions by centrifugation. Briefly, it was reasoned that the dispersion may consist of a dense DNA rich fraction and vesicles with little associated DNA that make a dominant contribution to the narrow 3 1 P NMR signal. As shown in Figure 2.10, centrifugation of the pCMVPgal containing complexes at 11,000 g for 15 min resulted in different amounts of complexes that can be pelleted depending on the charge ratio. The maximum amount of lipid and DNA in the pellet fraction appears in the region having 85 Figure 2.9 Fusogenic properties of plasmid DNA-cationic lipid complexes Fusion of pCMV5-DOTMA/DOPE (1:1) complexes with DOPS/DOPC (1:1) LUVs in 20 mM HEPES (pH 7.4) at 25 °C. The D O T M A concentration was 0.024 mM in each assay. Fusion assays were carried out as described in Materials and Methods. The charge ratio of each system is indicated and is defined as the moles of D O T M A to moles of phosphorus in pCMV5. System without pCMV5 is indicated by "n.a.". The presented data represent the average of three individual experiments. 120 §100 g 80 3 «, n.a. -20 0 30 60 90 120 150 180 210 240 Time (s) 86 Figure 2.10 Influence of charge ratios on the fractionation of DNA-cationic lipid complexes DNA and lipid recovery in supernatant and pellet fractions after centrifugation of DNA-cationic lipid complexes. Trace amounts of 3H-pCMVPgal and 1 4 C - C H E , and 20 pg pCMVPgal were used for both pCMVpgal-DOTMA/DOPE (A) and pCMVpgal-DOTMA/DOPC (B). DNA (circles) and lipid (squares) recovery in the supernatant (dotted lines) and the resuspended pellet fractions (solid lines) are shown. The charge ratio is defined as the moles of D O T M A to that of phosphorus in pCMVPgal. The average and standard deviation from three individual experiments are shown. 87 charge ratio 1.0. Contrary to expectations, there was no evidence for a DNA rich pellet; the lipid and DNA were separated in approximately equal proportions into the pellet and supernatant. It is therefore probable that the pellet and supernatant fractions differ in size rather than density, with the larger complexes contributing the pellet fraction. Notably, when DOPC was substituted for DOPE, the pellet fraction was increased to over 90 % of the lipid and plasmid as compared to 50 % for the DOPE containing system. The 3 1 P NMR characteristics of the pellet and supernatant at a charge ratio of 1.0 for both DOPE and DOPC containing complexes are shown in Figure 2.11. The 31 "31 supernatant P NMR peak remains narrow in both cases. As may be expected, the P N M R resonance for the pellet fractions are broader, however the resonance observed for the pellet obtained from the DOPE containing system is again substantially narrower than the bilayer lineshape derived from the DOPC containing system. Similar 3 1 P NMR phenomena were observed for both pCMV5 and pCMVPgal containing complexes (data not shown). 2.3.7 The transfection potency of pCMVpgal-DOTMA/DOPE (1:1) complexes correlate with the pellet fraction The relation between the structural and motional properties of the DNA-cationic lipid complexes and their ability to transfect target cells is of obvious importance. The transfection of B H K cells employing pCMVPgal-DOTMA/DOPE (1:1) complexes at various charge ratios is illustrated in Figure 2.12. It may be observed that maximum transfection is observed at a charge ratio of 1.0, in agreement with previous studies (Gao and Huang, 1991; Feigner et al., 1994). No transfection was observed at charge ratios 88 31 Figure 2.11 P NMR of the fractionated DNA-cationic lipid complexes 31 P NMR spectra of pCMV5-cationic lipid complexes at a charge ratio of 1.0 in HEPES buffer (20 mM; pH 7.4) at 25 °C. 3 1 P NMR spectra are shown for the supernatant (A, C) and the pellet fractions (B, D) of pCMV5-DOTMA/DOPE complexes (A, B) and pCMV5-DOTMA/DOPC complexes (C, D) after centrifugation for 15 min at 11,000 g. The charge ratio is defined as the ratio of moles of D O T M A to moles of phosphorus in pCMV5. 100 0 -100 100 o ^too PPM PPM 89 Figure 2.12 Transfection properties of complexes formed at different charge ratios Transfection of B H K cells by pCMVPgal-cationic lipid complexes at various charge ratios. Triplicates of the transfection results for pCMVPgal-DOTMA/DOPE (1:1) complexes (solid bars) and pCMVPgal-DOTMA/DOPC (1:1) complexes (open bars) on B H K cells using the p-galactosidase assay as described in Materials and Methods are shown. The charge ratio is defined as the moles of D O T M A to moles of phosphorus in pCMVPgal. Transfection by "naked" pCMVpgal is indicated in panel D. The average and standard deviation from triplicates of data are shown. 90 below 1.0; and the levels of transfection decrease to background levels at charge ratios greater than 4.0. The importance of DOPE as a "helper" lipid is clear, again in agreement with previous studies (Feigner et al., 1987; Feigner et al., 1994; Farhood et al., 1995), as no significant transfection is observed when DOPC is substituted for DOPE. No transfection was observed in the absence of plasmid or lipid. The next set of transfection studies examined the relative transfection potencies of the pellet and supernatant fractions. Remarkably, as shown in Figure 2.13, the pellet fractions containing large complexes exhibited significantly greater transfection potency than the unfractionated complex or the supernatant fractions. Interestingly, the reduction in transfection potency of the unfractionated complexes at higher charge ratios (Figure 2.12) corresponds approximately to the amount of material that can be pelleted by centrifugation (Figure 2.10). 91 Figure 2.13 Transfection properties of the fractionated DNA-cationic lipid complexes Transfection of B H K cells by the fractionated and unfractionated DNA-cationic l ipid complexes obtained by centrifugation. Triplicates of the transfection results for the unfractionated p C M V p g a l - D O T M A / D O P E (1:1) complexes (solid bars), the pellet fractions (gradient bars), and the supernatant fractions (open bars) at charge ratios (+/-) of 1.0, 1.5, and 2.0 are indicated respectively, using the P-galactosidase assay as described in Materials and Methods. N o transfection was observed in the presence o f "naked" p C M V P g a l . The average and standard deviation from triplicates of data are shown. 1.0 1.5 2.0 Charge Ratio (+/-) 92 2.4 DISCUSSION This investigation was focused on characterizing the structural, motional, and fusogenic properties of DOTMA/DOPE systems in isolation and in the presence of plasmid DNA. There are three major points of interest in the results obtained. These concern the ability of plasmid DNA and other multivalent anions to induce fusion between cationic LUVs, the structural and motional features of the complexes formed, and the relation between these features and transfection potential. We discuss these areas in turn. The ability to form DOTMA/DOPE LUVs relies on the ability of the cationic lipid D O T M A to stabilize DOPE into a bilayer organization, which is similar to the ability of anionic phospholipids, such as phosphatidylserine (PS), to stabilize DOPE into the bilayer phase (Cullis and de Kruijff, 1979). Further, the ability of polyanions such as plasmid DNA to trigger fusion between DOTMA/DOPE (1:1) LUVs is analogous to the ability of mono and divalent cations to trigger fusion between PS/PE vesicles as demonstrated extensively elsewhere (Tilcock and Cullis, 1981; Bally et al., 1983; Hope et al., 1983; Duzgune§ and Papahadjopoulos, 1983; Duzgiine§ et al., 1987). The mechanism whereby divalent cations such as C a 2 + can induce fusion in these systems has been shown to arise primarily from effects related to neutralization of the surface charge due to the anionic lipid rather than lateral segregation of the anionic species into local domains; although lateral segregation can occur in certain situations (Tilcock et al., 1984). Neutralization of the surface charge leads to a reduced effective head group size of the charged lipid species and correspondingly reduced ability to stabilize the bilayer organization. In addition, this reduced surface charge leads to reduced intervesicular 93 electrostatic repulsion, thus promoting the aggregation step required for fusion. It is likely, but not proven, that the ability of plasmid to induce fusion in the DOTMA/DOPE LUVs investigated here is also due to charge neutralization effects, as there is no evidence of lateral segregation of DOTMA. Two observations support this. First, the only situation giving rise to lateral phase separation of PS in PS/PE systems arises due to segregation of PS-Ca complexes into crystalline domains. There was no evidence as detected by freeze fracture for the presence of crystalline domains, which give rise to characteristic freeze fracture morphology. Second, the 3 1 P NMR spectra arising from the DOTMA/DOPE vesicles in the presence of plasmid are not consistent with lateral segregation of the D O T M A component. Specifically, if the D O T M A component was appreciably laterally segregated, DOPE would be expected to revert to the Hn phase organization it adopts in isolation at high plasmid levels. This is not observed (Figure 2.3A). It should be noted that this behavior contrasts with the behavior of the DOTMA/DOPC systems in the presence of plasmid DNA. In this case, the addition of plasmid results in DOPC in the bilayer organization it adopts in isolation. This is fully consistent with sequestration of the D O T M A by the plasmid. This is also consistent with recent work by Mitrakos and Macdonald (1996) who describe the ability of polyadenylic acid to sequester cationic lipids in mixtures with PC. Lipid mixing studies characterizing fusion of cationic vesicles induced by monovalent and multivalent anions have been previously reported (Rupert et al., 1985; Stamatatos et al., 1988; Duzgunes et al., 1989). This includes oligonucleotides (Jaaskelainen et al., 1994) as well as genomic DNA (Gershon et al., 1993). The studies presented here allow a comparison of the potency of plasmid DNA with anions of 94 different valency. The high potency of pCMV5 plasmid DNA for fusing DOTMA/DOPE LUVs as compared to chloride or citrate anions can be attributed to the much tighter binding expected for multivalent anions to cationic surfaces, as well as an ability of the DNA polymer to crosslink vesicles, thus directly promoting aggregation. This aggregation potential is observed at low plasmid levels in DOTMA/DOPE systems (Figure 2.4B) where the formation of small clusters precedes the formation of highly fused complexes. Lipid mixing studies demonstrating fusion between cationic and anionic vesicles have also been previously reported (Stamatatos et al., 1988; Duzgune§ et al., 1989; Bailey and Cullis, 1997). The main point of the studies presented here concerns the ability of high levels of plasmid DNA to inhibit fusion between DOTMA/DOPE systems and anionic (DOPS/DOPC) systems. This is fully consistent with charge repulsion effects inhibiting interactions between the DNA-cationic lipid complexes and the negatively charged vesicles. Related effects have been observed for oligonucleotide-cationic lipid complexes (Jaaskelainen et al., 1994). Such effects are also consistent with the inhibition of transfection at high plasmid DNA to cationic lipid levels (Figure 2.12), which presumably reflect reduced association with target cells. The nature of the complexes formed on addition of plasmid DNA to DOTMA/DOPE vesicles, and the relation between this structure and transfection potential is of importance. Previous studies on the relation between the structure of cationic lipid-plasmid DNA complexes and transfection potential have focused primarily on morphology as detected by electron microscopy techniques. The work of Sternberg et al. (1994) suggests that transfection potency correlates with the presence of long strands of DNA encapsulated in tubules of lipid. Such structures were not observed in the freeze 95 fracture studies performed here, although lipidic particle structures associated with non-bilayer lipid structures were observed, as also reported by Sternberg et al. (1994). As indicated below, the presence of non-bilayer lipid structure is consistent with the 3 1 P N M R studies. The most striking result of the studies presented here is that the more potent transfection systems contain lipid in large structures that allow significant isotropic motional averaging. The non-fractionated DOTMA/DOPE complexes formed with plasmid DNA exhibit a narrow isotropic 3 I P NMR signal, whereas considerably broader bilayer 3 1 P NMR spectra are observed for the DOTMA/DOPC systems in the presence of similar levels of plasmid. The isotropic signal observed for the non-fractionated plasmid-DOTMA/DOPE complexes with high transfection ability cannot be attributed to small vesicular structures, as the fusion, light scattering and freeze fracture studies reveal the presence of large, highly fused, lipid aggregates. In the case of the system fractionated by centrifugation, the 3 1 P NMR resonance observed for the DOPE containing system is broader, but remains significantly narrower than non-transfecting counterparts containing DOPC. Isotropic 3 1 P N M R signals are characteristic of lipid dispersions which form non-bilayer structures such as cubic or other inverted phases (Cullis and de Kruijff, 1979; Ellens et al., 1989; Lindblom and Rilfors, 1989), and variants such as interlamellar attachment sites may play direct roles in membrane fusion phenomena (Siegel et al., 1989). Such lipid organization could promote fusion or membrane destabilization after interaction with target cell membranes. Available evidence suggests that destabilization of the endosomal membrane is a dominant route for cationic liposome mediated transfection (Farhood et al., 1995; Wrobel and Collins, 1995; Zabner et al., 1995; Friend 96 et al., 1996; Xu and Szoka, 1996). The results presented here suggest that non-bilayer lipid structures could play a direct role in this process, thus facilitating the transfection event. 97 CHAPTER 3 STABILIZED PLASMID-LIPID PARTICLES: FACTORS INFLUENCING DNA ENTRAPMENT AND TRANSFECTION PROPERTIES 3.1 INTRODUCTION Most currently available systems for in vivo gene therapy are of limited utility for systemic applications such as treatment of inflammatory or metastatic diseases. Viral systems, for example, are rapidly cleared from the circulation, limiting transfection sites following intravenous injection to organs of the reticuloendothelial system (RES) such as the lung, liver or spleen. Alternatively, non-viral delivery systems such as plasmid DNA-cationic lipid complexes are rapidly cleared from the circulation due to their large size and positive charge, again limiting transfection sites to RES organs (Zhu et al , 1993; Mahato et al., 1995; Solodin et al., 1995; Litzinger et al., 1996; Liu et al , 1997; Templeton et al., 1997). In this regard it has recently been shown that plasmid DNA can be encapsulated in stabilized plasmid-lipid particles (SPLP) employing a detergent dialysis technique (Wheeler et al., 1998). These particles are small (diameter approximately 70 nm), protect encapsulated plasmid from degradation by serum nucleases, and can exhibit extended circulation times in vivo resulting in preferential accumulation in disease sites such as tumour sites. This has been shown to lead to transfection of distal tumour tissue following intravenous injection of SPLP as demonstrated by expression of a marker enzyme (R. Graham, personal communication). 98 It is of obvious interest to characterize factors that could influence the transfection properties of SPLP in vitro, with the aim of improving SPLP-mediated in vivo expression of therapeutic transgenes. Such factors include the cationic lipid contained in the SPLP and the poly(ethylene glycol) (PEG) coating surrounding the SPLP. For example, plasmid DNA-cationic lipid complexes which contain different cationic lipids can exhibit markedly different transfection properties both in vitro and in vivo (Gao & Huang, 1991; Rose et al., 1991; Jarnagin et al., 1992; Feigner et al , 1994; Egilmez et al., 1996; Templeton et al., 1997). Alternatively, it has been shown that the presence of PEG coatings on vesicles can dramatically inhibit intervesicular contact and fusion (Holland et al., 1996b); and the presence of PEG-PE in DNA-cationic lipid complexes can reduce in vivo transfection activity (Hong et al , 1997). In this work we investigate the influence of different species of cationic lipid and PEG coatings on the in vitro transfection properties of SPLP. It is shown that whereas the SPLP formulation process is relatively independent of the (monovalent) cationic lipid species employed, the highest levels of expression are observed for A^-[2,3-(dioleyloxy)propyl]-A^,Ar-dimethyl-A^-cyanomethylammonium chloride (DODMA-AN). Alternatively, incorporation of shorter acyl groups in the ceramide which "anchors" the PEG to the SPLP surface dramatically improves transfection levels. Finally, it is shown that low levels of cellular uptake can be a dominant parameter modulating the transfection potential of SPLP. 99 3.2 MATERIALS AND METHODS 3.2.1 Lipids and Chemicals ^A^-dioleyl-^A^-dimethylammonium chloride (DODAC), AyV-distearyl-AyV-dimethylammonium chloride (DSDAC), 7Y-[2,3-(dioleyloxy)propyl]-7Y,/Y-dimethyl-7Y-cyanomethylamrnonium chloride (DODMA-AN), 3-fi-[N-(N" ,AT'-dimethylaminoethyl)carbamoyl]-cholesterol (DC-CHOL) were obtained from Dr. S. Ansell of Inex Pharmaceuticals Corporation (Burnaby, British Columbia). l-0-(2'-(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-A/-octanoylsphingosine (PEG-CerCg), 1 -O-(2'-(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-A^-myristoylsphingosine (PEG-CerCi4), l-0-(2'-(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-A^-arachidoylsphingosine (PEG-CerC2o), l-0-(2'-(co-methoxypolyethyleneglycol(750))succinoyl)-2-V-myristoylsphingosine (PEG75o-CerCi4), l-0-(2,-(co-methoxypolyethyleneglycol(5ooo))succinoyl)-2-7Y-myristoylsphingosine (PEG5ooo-CerCi4) were obtained from Z. Wang of Inex, and 3H-labeled p C M V C A T plasmid were obtained from A. Annuar of Inex Pharmaceuticals Corporation. Spectra/Por 2 molecularporous membrane tubing (MW 12,000-14,000) was purchased from V W R Scientific (Edmonton, Alberta). Sodium acetate, sodium phosphate (Na2HP04), and sucrose were obtained from Fisher Scientific (Fair Lawn, NJ). N-octyl-/?-D-glucopyranoside (OGP), diethylaminoethyl (DEAE) Sepharose CL-6B anion exchanger, and Sepharose CL-4B resins were obtained from Sigma Chemical Co. (St. Louis, MO). Aquacide II was purchased from Calbiochem (La Jolla, CA). Xhol, Hindlll, and BamHI restriction endonuclease and ribonuclease I "A" from bovine pancreas (RNase) were 100 purchased from Pharmacia Biotech (Uppsala, Sweden). All other lipids and chemicals were described in a previous section (2.2.1). 3.2.2 Plasmid p C M V C A T plasmid, initially obtained from Inex Pharmaceuticals Corporation, and pCMVPgal plasmid, initially purchased from Clonetech, were amplified in Escherichia coli (DH5a) with the selection of resistance to ampicillin, and were isolated by alkali lysis and purified by PEG precipitation as described in a previous section (2.2.3). The purity of p C M V C A T and pCMVPgal was confirmed by 1 % agarose gel electrophoresis with restriction endonuclease Xhol or Hindlll digest, respectively. 3.2.3 Preparation of plasmid DNA-cationic lipid complexes Plasmid DNA-cationic lipid complexes were formed by incubating appropriate amounts of preformed DOTMA/DOPE (1:1) LUVs with pCMVpgal to obtain the desired charge ratio (positive-to-negative) of 1.0 in 100 uL distilled water. The resulting mixture was incubated at room temperature for 20 to 30 min, and was then mixed with an equal volume of culture media (see below). 3.2.4 Preparation of stabilized plasmid-lipid particles For each preparation, appropriate amounts of cationic lipid dissolved in chloroform were dried under a stream of nitrogen gas. Similarly, appropriate amounts of DOPE, PEG-ceramide and trace amounts of 1 4 C - C H E (as the lipid marker) were mixed in chloroform and dried. Residual solvent was removed under high vacuum for 2 hours. The resulting 101 lipid films were hydrated separately. n-Octyl-p-D-glucopyranoside (OGP, 50 uL of a 1.0 M solution) was added to the dried cationic lipid followed by brief vortexing. Subsequently 200 uL of an aqueous solution containing appropriate amounts of NaCl, p C M V C A T and trace amounts of 3 H - p C M V C A T was added. Unless indicated otherwise, 25 L i g p C M V C A T and 5.0 umol total lipids in 0.5 mL total volume were used for each preparation in the entrapment studies. After thorough mixing, the clear homogenous mixture was incubated at room temperature for 30 min. A final concentration of 200 mM OGP in 150 mM NaCl was used for each preparation. The lipid-containing medium was made by adding 250 uL of 200 mM OGP in 150 mM NaCl to the dried DOPE/PEG-Cer/ 1 4 C-CHE. After mixing and incubating at room temperature for 30 min, a clear homogenous mixture was obtained. The two mixtures were then mixed together and transferred into a 6 cm Spectra/Por 2 molecularporous membrane tubing (MW 12,000-14,000) for dialysis for 40 h against two changes of a 150 mM NaCl, 5 mM HEPES (pH 7.4) buffer. The SPLP thus formed were then purified by D E A E anion exchange chromatography and sucrose density gradient centrifugation, and then characterized with respect to plasmid entrapment and size (see below). All experiments involved triplicate samples. For the transfection studies, samples of SPLP containing pCMVPgal were prepared similarly with the exception that 400 p,g pCMVPgal, 10 umol total lipids, and a total volume of 1.0 mL were used initially. 102 3.2.5 Quantification of DNA entrapment and lipid recovery using anion exchange column chromatography A column chromatography procedure employing DEAE-Sepharose CL-6B to remove free DNA was utilized. The average of two 40 pL aliquots of the SPLP dialysate radioactivity was used as a reference for 3 H and 1 4 C radioactivity. The SPLP solution (100 pL of the dialysate) was applied to the DEAE-Sepharose column (diameter 1.0 cm, height 1.5 cm) and eluted using 150 mM NaCl, 20 mM HEPES (pH 7.4) buffer. Six fractions of 10 droplets each were collected in scintillation vials and were counted for 3 H and 1 4 C radioactivity. The percentage of recovery was obtained by comparing the total eluant radioactivity with the reference radioactivity after background correction. Thus the H recovery represents the fraction of DNA associated with the SPLP and sequestered from the anion exchanger, whereas the 1 4 C recovery represents the fraction of lipid in the void volume. Free plasmid DNA bound to the column could be washed out using 10 mL of a 5.0 M NaCl solution. A Beckman LS3801 scintillation counter was used for all radioactivity measurements. 3.2.6 Purification of SPLP using sucrose density gradient centrifugation All samples of SPLP used for transfection were further purified using sucrose density gradient centrifugation. SPLP were prepared as outlined above with 400 pg pCMV(3gal and 10 pmol total lipids in a total volume of 1.0 mL. Free DNA was removed by passing through a D E A E column. The diluted eluant (3 mL) was transferred to a dialysis bag and briefly dried by placing Aquacide II around the bag. After the desired volume was reached, the contents were dialyzed overnight in 150 mM NaCl, 5 mM HEPES (pH 7.4) 103 buffer. The concentrated eluant (800 uL) was then subjected to sucrose density gradient centrifugation (160,000 g for 2.5 h). The gradient was formed by loading 3.67 m L each of 1.0 %, 2.5 %, and 10 % sucrose in 150 m M N a C l , 20 m M H E P E S , p H 7.4 buffer into a centrifuge tube (Beckman Ultra-Clear Tubes) using a drawn out glass pipette. The crude S P L P was then applied to the top of the gradient and centrifuged using a swinging bucket rotor (SW-41Ti) in an ultracentrifuge (Beckman L2-65B). Following centrifugation, a band of concentrated S P L P with high D N A content was observed at the interface between the 2.5 % and 10 % sucrose gradient levels. The fractionation profile was achieved by removing 250 u L fractions from the top of the gradient, and these fractions were counted for 3 H and I 4 C radioactivity. A n aliquot (100 uL) of the sample before density gradient separation was also counted as reference. For the transfection studies, the band corresponding to S P L P containing high D N A content was isolated using needle suction. The resulting purified S P L P were concentrated by Aquacide II treatment as outlined above, and then dialyzed against 150 m M N a C l , 5 m M H E P E S (pH 7.4) with one change of buffer. Quantification of D N A and lipid was performed by comparing the 3 H and 1 4 C radioactivity of the purified S P L P against the radioactivity of crude S P L P before sucrose density gradient. The overall recoveries of D N A and l ipid were computed by multiplying the recovery from the D E A E column by the recovery from sucrose density gradient. A s shown in Figure 3.1, clear separation of the S P L P containing plasmid and the l ipid component containing little or no D N A was achieved. The lipid and D N A recovery from the D E A E column, from sucrose density gradient centrifugation, and the combined recovery from both purification processes, are outlined in Table 5 for S P L P formed with 104 Figure 3.1 Purification of stabilized plasmid-lipid particles SPLP composed of DOPE/DOTMA/PEG-CerCg (73:7:20; mol ratios) initially containing 400 pg pCMVPgal and 10 pmol total lipid were prepared. Separation of SPLP containing plasmid DNA from empty vesicles was achieved using a sucrose density step-gradient (1.0 %, 2.5 %, and 10 %) centrifuged at 160,000 g for 2.5 h. Fractions were counted for radioactivity starting from the top of the gradient. 3 H pCMVPgal (closed circles) and 1 4 C -C H E (open circles) were used as plasmid and lipid markers, respectively. 1 8 15 22 29 36 43 Fraction Number 105 in 3 a o -*»-CJ a u •*•> «s - J c» o a CJ u 3 OH o ^ o a o £ o o o -a a I1 o CU DJD « a C J s-V cn O i . cj 9 C/3 Q c _« "3 S S-O -a a. 2 r - l 0 0 VO CN C 0 0 I T ) ON r--r t o CN CN u U ii U 6 w e< u Q O o Q o VO VO Tt o • i-H bi) a CD O <D T3 cd i-i b o C/3 c u T3 CD 1/3 o l-l o cs c/3 l-l u P3 T 3 <D g '3 -*-» X> O T3 a, Q o t/3 <D • t—I I i > o o a <D H a o id bO i i Ci <D o c! cd I-I bO Cl CD T3 <D C/3 o l-l o 3 o o W < Q o l-l g -4 -> o !& T 3 o > o o <u l-l o x CD o o Cl o c/3 CD Cd bO > o P H b o = u I-i <D C H C/3 T 3 o CD a CD C/3 1 ) S i C M CD I i O C/3 CD 3 > CD X3 H 106 either D O T M A or DODAC as the cationic lipid. These purified SPLP had particle sizes of 110 ± 20 nm as measured by QELS. 3.2.7 Size determination of SPLP employing quasi-elastic light scattering Purified SPLP containing high plasmid content were transferred into a 6 x 50 mm borosilicate glass tube and placed in a Nicomp Model 270 submicron particle sizer for size analysis, using particle mode in QELS. 3.2.8 Transfection studies employing BHK cells All transfection procedures were carried out in a laminar flow hood (Forma Scientific). Purified SPLP were prepared as described above. Unless indicated otherwise, SPLP samples containing 2.0 pg pCMVPgal, and plasmid DNA-cationic lipid complexes containing 0.5 pg were used. Complexes (charge ratio of 1.0) were formed by incubating DOTMA/DOPE (1:1) vesicles with appropriate amounts of pCMVPgal for 30 min before transfection, as outlined in section 3.2.3. All transfection studies were performed in triplicate as outlined in section 2.2.9. For each well containing the cells, appropriate amounts of complexes or SPLP were diluted with DMEM/FBS, and aliquots of 100 pL were used for transfection at 37 °C, 5 % CO2 with incubation time of 24 or 4 h, respectively. The cells were then lysed and freeze-thawed (section 2.2.9). After thawing, aliquots of 10 pL of the lysis buffer was transferred to another 96 wells plate for protein analysis using the B C A assay (from Pierce). The remaining samples were assayed for P-galactosidase activity (section 2.2.9). For the protein assay, 50 pL of BSA standard (0 -20 pg) was prepared by serial dilution in lysis buffer. An additional 40 pL of lysis buffer 107 was added to the 10 p L sample aliquot. B C A reaction mixture was prepared according to established protocols from Pierce (Rockford, IL) prior to the assay, and 100 p L of this mixture was added to each well , including the protein standards. The plate was incubated at 37 °C for 2 h or until the color was developed. The amounts of P-galactosidase and protein were quantified against the standard, after volume adjustment, and the P-galactosidase activity was expressed as milliunit of P-galactosidase per mg of protein. A l l absorbance readings were measured at 540 nm using a Microplate Autoreader EL-309 (Bio-Tek Instruments). For the transfection of S P L P formed with different PEG-Cer , 1 x 10 5 B H K cells were plated in a 24-well plate. Aliquots of 60 p L and 10 p L were used for the B S A and B C A assay, respectively. 3.2.9 Cellular uptake studies of plasmid DNA B H K cells were plated at a density of 5 x 10 5 cells per 25 c m 2 T-flask the day before transfection. For each transfection, DNA-cationic l ipid complexes containing 2 pg p C M V p g a l at a charge ratio of 1.0 were formed by incubating D O T M A / D O P E (1:1) L U V s with p C M V P g a l for 20 to 30 min at room temperature before transfection. Purified S P L P samples (DOTMA/DOPE/PEG 2 ooo -CerC 8 , 7:73:20) containing 2 pg p C M V p g a l were also used for each transfection. Both plasmid DNA-cationic l ipid complexes and S P L P samples were made up to a final volume of 2 m L with D M E M containing 10 % F B S before applying to B H K cells. For the cells transfected with complexes, the transfection medium was replaced with complete D M E M after the 4 h incubation time point. Cells transfected with the S P L P samples were incubated in the transfection medium until the specific time point. A l l transfection studies were performed in triplicate. 108 The cellular uptake kinetics of the plasmid DNA were analyzed by terminating the transfection process at 4, 8, 24, and 48 h. At each time point, the transfection medium was removed. The cells were washed twice with PBS, and then treated with trypsin-E D T A (0.05 % trypsin, 0.53 mM EDTA-4Na from GibcoBRL). The cells were then washed with an isotonic buffer (250 mM sucrose, 50 mM HEPES, pH 7.2, 3 mM MgCl 2) and were centrifuged at 2,000 rpm for 2 min in a Sorvall M C 12 V centrifuge. The resulting cell pellets were resuspended in the isotonic buffer, and the number of cells was counted using a hemacytometer. Then, the cells were centrifuged at 2,000 rpm for 2 min, and the pellets were treated with the lysis buffer (10 mM Tris, pH 7.5, 0.5 % SDS, 1 mM EDTA) containing Pronase E (1 mg/mL) at 37 °C overnight. 3.2.10 Southern blot analysis of delivered plasmid DNA Genomic DNA was isolated from the BHK cells transfected with DNA-cationic lipid complexes and SPLP (Sambrook et al., 1989). Briefly, the cell lysates were extracted twice with phenol/chloroform (1:1), then the DNA was precipitated with 95 % ethanol and resuspended in T E at 200 pL per 1 x 106 cells. The recovery of total genomic DNA was determined by measuring the absorbance at 260 nm of the resuspended samples. Genomic DNA (5 pg) from each sample was loaded into a 1 % agarose gel with a set of plasmid DNA standards (0 to 5 ng). After size fractionation, the agarose gel was incubated in a denaturing buffer (1.5 M NaCl, 0.5 N NaOH) for 1 h, and then in a neutralizing solution (1.5 M NaCl, 1 M Tris, pH 7.4) for 45 min. The DNA fragments were transferred to a nylon membrane by capillary blotting overnight with 3 M NaCl, 0.3 M sodium citrate (pH 7.0). The nylon membrane was then baked at 80 °C for 1 h prior to 109 the hybridization procedure. A 32P-labeled plasmid DNA probe was prepared using the T 7 QuickPrime™ Kit (Pharmacia Biotech) with BamHI cut pCMVpgal. This probe was then added to the DNA blot and allowed to hybridize overnight at 68 °C. The blot was washed 3 times with 300 mM NaCl, 30 mM sodium citrate (pH 7.0) containing 0.1 % SDS and once with 30 mM NaCl, 3 mM sodium citrate (pH 7.0) containing 0.1 % SDS. The blot was then exposed for 2 to 4 h on a Phospholmager screen and subsequently scanned (Molecular Dynamics - PhosphoImager™SI). The amount of plasmid DNA taken up into cells was normalized by dividing the total plasmid DNA (pg) recovered by the total genomic DNA (pg). 110 3.3 RESULTS 3.3.1 Influence of cationic lipid species on formation of SPLP Previous work has shown that incubation of plasmid DNA with the lipid mixture DOPE, PEG2ooo-CerC2o and the cationic lipid DODAC (84:10:6; molar ratios) in the presence of OGP followed by dialysis results in the formation of SPLP which are capable of transfection (Wheeler et al., 1998). The low levels of DODAC in SPLP are required to achieve plasmid loading. It is of interest to determine whether SPLP can be formed using other cationic lipids, and whether this influences transfection potency. Here we characterize SPLP formation and plasmid entrapment achieved using D O T M A , D O D M A - A N , DSDAC, DODAC, and DC-CHOL (for structures see Figure 3.2) using a total of 5.0 pmol lipid and 25 pg p C M V C A T plasmid. In these systems the PEG-CerCn content was held constant at 10 mol % of total lipid, and the cationic lipid content varied over the 0 - 2 0 mol % range. Plasmid entrapment was assayed employing the D E A E anion exchange column procedure detailed under Materials and Methods. As shown in Figure 3.3, optimum entrapment levels of approximately 60 % were achieved for each of the cationic lipids used, the only difference being that this maximum entrapment was observed at slightly different cationic lipid content depending on the cationic lipid species. The optimal lipid composition (DOPE/cationic lipid/PEG-CerCu; molar ratios) for plasmid encapsulation was 83:7:10 for D O T M A and D O D M A - A N , 82.5:7.5:10 for DSDAC and DODAC, and 81:9:10 for DC-CHOL. These SPLP exhibited particle sizes of 80 ± 20 nm as measured by QELS in the particle mode. Ill Figure 3.2 Structures of the cationic lipids used in this study D O T M A DODAC Cl 112 Figure 3.3 Influence of cationic lipids on plasmid encapsulation in SPLP Plasmid encapsulation efficiency for the SPLP detergent dialysis procedure as a function of cationic lipid content for a variety of cationic lipids: (A) D O T M A (•), D O D M A - A N (*), (B) DSDAC (A), DODAC (•), and (C) DC-CHOL (•). SPLP were prepared as described in Materials and Methods, employing 25 u.g p C M V C A T plasmid with 5.0 umol total lipid consisting of 10 mol % PEG-CerCn, x mol % cationic lipid, and 90-x mol % DOPE. Encapsulation was assayed by measuring DNA recovery after passage of the dialysate through a D E A E Sepharose CL-6B column (see Materials and Methods). The average and standard deviation from three individual experiments are shown. 100 ^r^VT'&i T ? j ' l l l l l l l l l | l l l l l l l l l | l l l l l l l l l 5 10 15 20 Cationic lipid content (%) 100 5 10 15 Cationic lipid content (%) 20 100 o to CU < 5 10 15 Cationic lipid content (%) 20 113 3.3.2 Influence of the PEG polymer anchor on formation of SPLP The poly(ethylene glycol) (PEG) coating of SPLP is likely to inhibit association with cells, thus reducing transfection efficiency. Previous work has shown that SPLP constructed from PEG-CerC2o did not result in appreciable transfection in vitro, whereas limited transfection was observed for SPLP containing PEG-CerCi4 (Wheeler et al., 1998). This was attributed to an ability of the PEG-CerCi 4 to dissociate from the SPLP surface. Here, this work is extended to examine the formulation and transfection properties of SPLP containing PEG2000 polymers linked to ceramide anchors containing octanoyl acyl groups (PEG-CerC8). Results for SPLP containing PEG-CerCi 4 and PEG-CerC2o are also presented for comparison (for structures see Figure 3.4). Preliminary experiments suggested that more than 10 mol % of the PEG-CerCg was required to achieve optimal plasmid entrapment and, therefore, the entrapment profile was generated as a function of PEG-ceramide content rather than cationic lipid content. The detergent dialysis protocol was then applied to systems containing 25 pg p C M V C A T and 5.0 pmol total lipids where the cationic lipid content was maintained at 7.5 mol % DODAC. As shown in Figure 3.5, maximum entrapment levels are observed at approximately 20 mol % PEG-CerCg, whereas the maximum plasmid entrapment for the PEG-CerCi 4 and PEG-CerC2o systems are in the range of 10 - 12 mol % of the PEG-ceramide. 3.3.3 Influence of the PEG anchor on transfection properties of SPLP The transfection properties of SPLP were investigated employing SPLP with entrapped pCMVPgal to allow a convenient assay for transfection. Further, the transfection protocol involved using purified SPLP, where the empty vesicles produced during the detergent 114 Figure 3.4 Structures of poly(ethylene glycol)-ceramides (PEG-Cer) used in this study CH3(OCH2CH2)nO O N ^(CH 2 ) X CH 3 P E G 2 0 0 0 CerC 8 n = 45; x = 6 P E G 2 0 0 0 C e r C 1 4 n = 45; x = 12 P E G 2 0 0 0 C e r C o n 20 n = 45; x = 18 P E G 7 5 0 C e r C 1 4 n = 17; x = 12 P E G 5 0 0 0 C - C 1 4 n = 114; x = 12 115 Figure 3.5 Influence of ceramide anchors on plasmid entrapment properties in SPLP Plasmid encapsulation efficiency for the SPLP detergent dialysis procedure utilizing (A) PEG-CerCg, (B) PEG-CerCi 4 and (C) PEG-CerC 2 0 . SPLP were prepared as described in Materials and Methods employing 25 Lig p C M V C A T formulated with 5.0 umol total lipids containing 7.5 mol % DODAC, x mol % PEG-Cer, and 92.5-x mol % DOPE. Encapsulation was assayed by measuring plasmid recovery after passage of the dialysate through a D E A E Sepharose CL-6B column. The average and standard deviations calculated from three individual experiments are shown. 100 PEG-CerC 2 0(mol %) 116 dialysis procedure were removed by density gradient centrifugation as detailed in Materials and Methods. The initial set of experiments was designed to ascertain appropriate transfection conditions for the SPLP (DOPE/DOTMA/PEG-CerC 8; 73:7:20; molar ratios) system. As shown in Figure 3.6A, protocols employing SPLP containing 0.5 pg pCMVPgal gave little or no transfection; however significant transfection was observed for SPLP containing 1.0 or 2.0 pg of plasmid DNA at transfection times of 24 h or longer. Thus, a 24 h incubation time and 2.0 pg plasmid DNA was utilized for the standardized SPLP transfection protocol in subsequent experiments. It is of interest to compare the transfection properties of SPLP with that achieved employing plasmid DNA-cationic lipid complexes. As shown in Figure 3.6B, transfection of B H K cells employing pCMVPgal-DOTMA/DOPE (1:1) complexes gave transfection levels which are approximately an order of magnitude higher than observed for the SPLP preparation. In addition, good transfection activity was observed at low (0.5 pg pCMVPgal) plasmid levels and at short (4 h) incubation times. In order to characterize the influence of the ceramide anchor on the transfection properties, SPLP containing pCMVPgal were generated using 400 pg pCMVPgal and 10 pmol lipid mixtures. Lipid compositions of DOPE/DOTMA/PEG-Cer (83.5:6.5:10; molar ratios) using the amide chain lengths of Cs, Cu, and C20 were prepared. These systems were then purified to remove empty vesicles employing the density centrifugation protocol. The transfection properties of the purified SPLP containing PEG-CerCg, PEG-CerCi4 and PEG-CerC2o in BHK cells under standard transfection conditions (2 pg pCMVpgal; 24 h incubation) are illustrated in Figure 3.7. A correlation between the transfection activities and the length of the ceramide anchor is observed. SPLP 117 Figure 3.6 Transfection properties of SPLP and plasmid DNA-cationic lipid complexes (A) P-galactosidase expression in BHK cells resulting from incubation with SPLP containing 0.25 Lig pCMVpgal (open bars), 1.0 u.g pCMVPgal (shaded bars), and 2.0 jag pCMVPgal (solid bars) for 4 and 24 h is shown. (B) P-galactosidase expression in B H K cells resulting from incubation with complexes containing 0.25 p,g pCMVPgal (open bar), 0.50 tig pCMVPgal (shaded bars), and 1.0 u.g pCMVpgal (solid bars) for 4 and 24 h. SPLP (DOPE/DOTMA/PEG-CerCg; 73:7:20; molar ratios) and plasmid DNA-cationic lipid complexes (DOTMA/DOPE (l:l)-pCMVPgal; +/- 1.0) were prepared from pCMVpgal as described in Materials and Methods. SPLP were purified employing the discontinuous sucrose density gradient centrifugation protocol. The average and standard deviation from triplicates are shown. 4 24 Transfection time (h) 1000 [ 24 Transfection time (h) 118 Figure 3.7 Transfection properties of SPLP composed of different PEG-ceramide anchors Transfection properties of SPLP containing PEG-CerCg, PEG-CerCn and PEG-CerC2o-B H K cells were transfected with SPLP composed of DOPE/DOTMA/PEG-Cer (83.5:6.5:10; mol ratios) and 2.0 pg pCMVPgal for 24 h as outlined in Materials and Methods. SPLP were purified employing the discontinuous sucrose density gradient centrifugation protocol. The average and standard deviation from triplicates are shown. 350 O) E 300 --'_ c 250 i -3 -E. 200 ; > 150 - . ^ 3 : < 100 --n ; 50 - . CQ j 0 • PEG-CerC8 PEG-CerC14 PEG-CerC20 119 formed with P E G - C e r C 8 give rise to 30 fold higher transfection activity than systems formed with P E G - C e r C i 4 , which in turn results in 8 fold higher transfection activity than S P L P formed with P E G - C e r C 2 0 . 3.3.4 Influence of cationic lipid species on transfection properties of SPLP The transfection properties of S P L P containing the different cationic lipids with 20 % PEG-CerCg are illustrated in Figure 3.8. The standard S P L P transfection protocol (2.0 u.g p C M V P g a l ; 24 h incubation time) was utilized. It may be observed that the inclusion of different cationic lipids in S P L P did lead to different transfection activity, leading to the transfection potency profde of D O D M A - A N > D O T M A > D O D A C > D S D A C > D C -C H O L . However in all cases, as also shown in Figure 3.8, S P L P are five to ten-fold less potent transfection agents than the plasmid DNA-cat ionic l ipid complexes ( D O T M A / D O P E (1 : l ) - p C M V P g a l ; 0.5 |xg D N A ; 24 h incubation time). 3.3.5 Influence of PEG polymer length on formation and transfection properties of SPLP A n alternative approach to improve the transfection potency of S P L P is to reduce the length of the P E G polymer associated with the ceramide anchor. This was investigated for PEG-CerCi4 molecules containing PEG750, PEG2000 and PEG5000. The plasmid entrapment properties of S P L P containing these PEG-ceramides are illustrated in Figure 3.9. Entrapment was measured as a function of D O D A C content for systems containing 25 jig p C M V C A T plasmid and 5 umol total l ipid with 10 mol % P E G 7 5 o - C e r C i 4 , PEG2ooo-CerCi4 or PEGsooo-CerCi 4. It may be observed that relatively lower entrapment 120 Figure 3.8 Comparison of the transfection properties of SPLP formed with different cationic lipids and DNA-cationic lipid complexes SPLP composed of cationic lipid (DODMA-AN and DOTMA, 7.0 mol %; D O D A C and DSDAC, 7.5 mol %; DC-CHOL, 9.0 mol %), DOPE, and 20 mol % PEG-CerCg containing 2.0 pg pCMVpgal, and pCMVPgal-DOTMA/DOPE (1:1) complexes (+/- = 1.0) containing 0.5 pg pCMVpgal were incubated with BHK cells and transfection assayed at 24 h. The average and standard deviation from triplicates are shown. 13 > "-4—» O CO "ro cn • CQ 121 Figure 3.9 Influence of PEG polymer lengths on plasmid entrapment in SPLP Plasmid encapsulation efficiency for the SPLP detergent dialysis process as a function of cationic lipid (DODAC) content utilizing PEG-ceramides which vary in the size of the PEG polymer incorporated. SPLP were prepared as described in Materials and Methods employing 25 pg p C M V C A T with 5.0 pmol total lipid consisting of 10 mol % PEG-Cer ((A) PEG7 5o-CerCi4, (B) PEG2ooo-CerCi4, and (C) PEG5ooo-CerCi4), x mol % DODAC, and 90-x mol % DOPE. Encapsulation was assayed by measuring plasmid ( 3H-pCMVCAT) recovery after passage of the dialysate through a D E A E Sepharose CL-6B column (see Materials and Methods). The average and standard deviation from three individual experiments are shown. 100 n 1 DODAC (mol %) 122 levels in the range o f 40 % are achieved for the PEG 7 5o-CerCi4 and PEGsooo-CerCn systems as compared to nearly 60 % for the PEG2ooo-CerCi4 containing system. Transfection studies were performed on S P L P formed initially with 400 u,g p C M V p g a l and 10 umol total lipids composed of D O P E , D O T M A , and P E G - C e r C i 4 (83.5:6.5:10; molar ratios) for all the P E G species. These preparations were then purified employing the density centrifugation procedure and used to transfect B H K cells according to the standard protocol. The transfection properties of these S P L P containing P E G 7 5 o - C e r C i 4 , PEG 2 0 oo -CerCi 4 and PEG 5 0 oo -CerCi 4 are illustrated in Figure 3.10. S P L P containing PEG750 have similar in vitro transfection potency than systems formed with PEG2000 or PEG5ooo-3.3.6 Comparison of intracellular delivery of plasmid by SPLP and complexes The results to this point indicate that S P L P containing D O D M A - A N , P E G anchors with shorter amide chains and P E G coatings composed of shorter P E G polymers result in improved transfection of B H K cells in vitro. However, in all cases the transfection levels achieved are substantially lower than those observed for plasmid DNA-cat ionic l ipid complexes. This may result from a reduced affinity of S P L P for cells due to the presence of the P E G coating on the S P L P and the much reduced positive charge on the S P L P as compared to complexes. Both of these effects may act to substantially reduce the amount of plasmid that is delivered to the cell. In order to determine whether this could account for the reduced transfection potency of S P L P as compared to complexes, the time dependent cellular uptake of p C M V P g a l in both lipid-based D N A carriers was investigated. S P L P were formed from 400 u.g p C M V P g a l and 10 umol total lipids 123 Figure 3.10 Influence of PEG polymer lengths on transfection properties of SPLP Transfection properties of SPLP containing PEG-ceramides in which the size of the PEG polymer is varied (PEG750, PEG2000 and PEGsooo)- p-galactosidase expression in BHK cells was assayed following incubation with SPLP containing 2.0 pg pCMVPgal for 24 h. SPLP (DOPE/DOTMA/PEG-CerCi 4 ; 83.5:6.5:10; mol ratios) containing pCMVpgal were prepared as indicated in Materials and Methods. SPLP were purified employing the discontinuous sucrose density gradient centrifugation protocol. The average and standard deviation from triplicates are shown. 124 composed of DOPE/DOTMA/PEG-CerC 8 (73:7:20; molar ratios) and were purified by density centrifugation. Purified SPLP samples (2 ug pCMVPgal) and DNA-cationic lipid complexes (DOTMA/DOPE (l:l)-pCMVPgal; charge ratio of 1.0; 2.0 ug pCMVPgal) were transfected as outlined in Materials and Methods. Plasmid DNA delivered by the complexes demonstrate rapid, high cellular uptake and subsequent degradation with maximum plasmid levels at 4 h; whereas SPLP yield maximum plasmid delivery at a 8 to 24 h incubation (Figure 3.11 A). It may be noted that the maximum levels of plasmid delivered by complexes is more than 30 times that delivered by SPLP. The integrity of the delivered plasmid DNA over time is illustrated in Figure 3.1 IB. Although the complexes delivered much more plasmid, this plasmid was readily degraded as compared to that delivered by the SPLP systems. This is indicated by the smears observed in the DNA delivered by the complexes as shown in the agarose gel electrophoretic pattern (Figure 3.1 IB). This observation indicates that plasmid DNA entrapped within the particles is not as susceptible to degradation by cellular enzymes as compared to plasmid DNA associated with complexes. 125 Figure 3.11 Southern blot analysis of plasmid DNA delivered by SPLP and plasmid DNA-cationic lipid complexes in BHK cells S P L P composed of D O P E / D O T M A / P E G - C e r C g (73:7:20; molar ratios) and complexes composed of D O T M A / D O P E ( l : l ) - p C M V p g a l (charge ratio of 1.0) were used. D N A transfection and cellular uptake studies were performed as described in Materials and Methods. Comparison of the cellular uptake of plasmid D N A using DNA-cat ionic l ipid complexes (•) and S P L P ( • ) is shown in panel A . Southern blot analysis of the integrity of the plasmid D N A delivered by the two lipid-based D N A carriers is shown in panel B . The average and standard deviation of triplicates are shown. Time (h) B Complexes SPLP c B 126 3.4 DISCUSSION This work was aimed at characterizing factors that influence the transfection potency of stabilized plasmid-lipid particles with the aim of improving transgene expression. It is shown that three factors that can modulate S P L P transfection efficacy are the species of cationic l ipid employed, the size of the P E G polymer coating the S P L P and the length of the acyl chain contained in the ceramide "anchor". These factors are discussed in turn, followed by a discussion of the implications of the observation that the low levels of S P L P uptake into cells may be the primary parameter limiting transgene expression. With regard to the influence of different cationic lipids, the results presented here demonstrate that plasmid encapsulation employing the detergent dialysis process is relatively independent of the species of monovalent cationic l ipid employed. Although it is difficult to discern any definite trends, D O D M A - A N and D O T M A appear to provide the maximum entrapment at the lowest cationic l ipid content (~ 7 %), followed by D O D A C and D S D A C (~ 7.5 %) and D C - C H O L (~ 9 %). Interestingly, S P L P composed of D O D M A - A N and D O T M A exhibit significantly higher transfection potencies than S P L P containing D O D A C , D S D A C or D C - C H O L (Figure 3.8). These results could be taken to suggest that cationic lipids with the greatest affinity for plasmid D N A as judged by entrapment under the conditions of detergent dialysis lead to the most potent transfection systems. In any event, it is clear that the species of cationic l ipid does influence the transfection capability of the resulting S P L P , with D O D M A - A N resulting in the highest transfection levels and D C - C H O L the lowest, with D O D A C and D S D A C giving rise to intermediate transfection levels. 127 The second factor which clearly plays a major role in modulating the transfection potency of SPLP is the length of the acyl chain contained in the hydrophobic ceramide group which anchors the PEG coating to the SPLP surface. Previous work has shown that inclusion of PEG-CerCn in SPLP results in enhanced expression in vitro as compared to SPLP containing PEG-CerC2o, however the levels of gene expression were low in all cases (Wheeler et al., 1998). The results presented here show that thirty-fold higher transfection levels can be achieved for SPLP containing PEG-CerCg as compared to PEG-CerCi4 (Figure 3.7). This improved transfection ability presumably reflects a faster leaving rate for the shorter chain PEG-ceramides from the SPLP surface in the sequence PEG-CerC 8 > PEG-CerCi 4 > PEG-CerC 2 0 , leaving the SPLP surface less shielded by PEG for the Cg containing system. This effect is fully consistent with previous work showing that fusion between LUVs can be inhibited by the presence of a PEG coating anchored to PE molecules, and that fusogenicity could be restored by using PE anchors with short acyl chains (Holland et al., 1996b). It is also consistent with the observation that the transfection potency of plasmid DNA-cationic lipid complexes is significantly reduced by the presence of PEG-PE (Hong et al., 1997). The ability of the PEG coating on SPLP to inhibit transfection can arise due to reduced binding and therefore reduced uptake into target cells, or reduced efficiency in fusing with the endosomal membrane in order to achieve intracellular delivery of the plasmid. The formulation properties of SPLP containing PEG-CerCg are clearly different from the properties of SPLP containing longer chain ceramides, in that optimum encapsulation is achieved at ~ 20 mol % PEG-CerCg as compared to ~ 10 mol % for SPLP containing PEG-CerCi 4 or PEG-CerC2o (Figure 3.5). There are two related effects 128 which could contribute to this phenomenon. First, it is likely that the critical micellar concentration (cmc) of PEG-CerC 8 is larger than that of the longer chain PEG-ceramides, leading to a higher proportion of the PEG-CerCg in micellar form in solution. Second, it is also possible that the partition coefficient of the shorter chain PEG-CerCg for the SPLP outer monolayer is reduced, again resulting in a higher aqueous concentration of the PEG-CerCg. The third parameter, the length of the PEG polymer, appears to influence the formulation properties of the SPLP more than the transfection potency. The transfection potency of SPLP containing P E G 7 5 0 , P E G 2 0 0 0 and P E G 5 0 0 0 coupled to CerCi 4 are similar (Figure 3.10), however the formulation properties differ significantly. In particular, the maximum efficiency for plasmid encapsulation that could be achieved for SPLP containing P E G 7 5 0 or PEG50oo was ~ 40 %, as compared to ~ 60 % for the P E G 2 0 0 0 system (Figure 3.9). Reduced plasmid entrapment for shorter PEG polymers would be expected due to the reduced steric stabilizing capacity of the shorter PEG polymers. The reason for the poorer entrapment for the SPLP containing PEGsooo-ceramide is not currently understood. It may result from a higher cmc of the PEGsooo-ceramide due to the larger size of the polar region, or to interference of the longer PEG polymer with the formation of lipid-coated plasmid which has been suggested to be an intermediate in the formation of SPLP (Wheeler et al., 1998). The fact that SPLP containing PEG-ceramides with shorter PEG polymers do not exhibit significantly higher transfection potencies, presumably reflects the fact that the presence of PEG-ceramides, which are sufficient to stabilize formation of the SPLP during the detergent dialysis process, are also sufficient to inhibit uptake into cells. 129 The final area of discussion concerns the observation that SPLP exhibit much lower levels of accumulation into target cells as compared to plasmid DNA-cationic lipid complexes, and that the plasmid delivered by SPLP remains intact in the cell for a longer time following cellular uptake. With regard to the first point, when BHK cells are presented with equivalent amounts of plasmid DNA in either the complex form or in SPLP (DOPE/DOTMA/PEG-CerCg; 73:7:20, molar ratios) form, the maximum amount of plasmid that is delivered into the cells by the SPLP is less than 3 % of the maximum amount delivered by the plasmid DNA-cationic lipid complexes. Thus even though the in vitro transfection potency of the complexes (DOTMA/DOPE (l:l)-pCMVpgal; charge ratio of 1.0; 2.0 pg pCMVpgal) is at least 10 fold higher than the SPLP (DOPE/DOTMA/PEG-CerCg; 73:7:20: 2 pg pCMVpgal; 24 h transfection), the reduced potency of the SPLP can be attributed almost entirely to low levels of cellular uptake. This argues strongly that SPLP exhibit an ability to transfect cells following uptake that is comparable to complexes, and that the most direct way to improve the transfection properties of SPLP is to enhance cellular uptake. This could be accomplished in a number of ways, including incorporation of external ligands to promote cell association and uptake. The second observation that plasmid delivered to cells by SPLP is broken down at a much slower rate than plasmid delivered in complexes presumably reflects the resistance of the SPLP particle to breakdown by intracellular factors. It also points out the potential for more stable particles such as SPLP to extend the duration of transfection resulting from transfection by non-viral gene delivery systems. It should be noted that the construction of SPLP, which are more stable, and which could therefore exhibit longer 130 intracellular residence times than the systems employed here, is straightforward. For example, substitution of saturated phosphatidylcholines for DOPE would be expected to significantly enhance SPLP stability and possibly promote extended gene expression. In summary, this investigation presents a study of factors that regulate the transfection potency of stabilized plasmid-lipid particles. It is shown that the transfection potency is sensitive to both the cationic lipid species and the species of PEG-ceramide employed to construct the SPLP. Improved transfection activity can be achieved by the use of the cationic lipid DODMA-AN, PEG-ceramides incorporating smaller PEG polymers and, most importantly, the use of PEG-ceramides containing shorter acyl groups in the ceramide anchor. Further, it is shown that the dominant factor leading to lower levels of transfection by SPLP is the reduction of SPLP accumulation by the target cells. These observations point the way to achieve plasmid delivery systems that exhibit enhanced levels of gene expression for in vivo gene therapy. 131 C H A P T E R 4 STABILIZED PLASMID-LIPID P A R T I C L E S : I N F L U E N C E O F PLASMID C O N F O R M A T I O N O N DNA E N T R A P M E N T A N D T R A N S F E C T I O N PROPERTIES 4.1 I N T R O D U C T I O N The previous chapter characterizes factors associated with the lipid component that influence the formation and transfection properties of SPLP. It is of obvious interest to examine other factors that could improve the formulation and the transfection properties of SPLP. Two drawbacks of the detergent dialysis protocol are that plasmid trapping efficiencies are limited to approximately 60% and that a large fraction of the lipid is in the form of empty vesicles. It is clearly desirable to generate SPLP formulations with higher trapping efficiencies where a greater proportion of the lipid present is actually associated with plasmid. In this work we examine how factors associated with the plasmid-to-lipid ratio and plasmid conformation can influence these properties. It is shown that high plasmid trapping efficiencies can be maintained to plasmid-to-lipid ratios as high as 0.076 (weight ratio) and that significantly higher trapping efficiencies can be achieved for linearized plasmid, as compared to plasmid in the supercoiled or relaxed circular conformations. However, formulations containing linearized plasmid were found to exhibit poor transfection properties. 132 4.2 MATERIALS AND METHODS Materials and methods not detailed here are already presented in sections 2.2 and 3.2. 4.2.1 Lipids, chemicals, and plasmid Cesium chloride (CsCl) and magnesium chloride (MgCb) were obtained from Fisher Scientific (Fair Lawn, NJ). Dithiothreitol and ethidium bromide were obtained from Sigma Chemical Co. (St. Louis, MO). Topoisomerase I was purchased from GibcoBRL (Burlington, Ontario). PicoGreen double stranded DNA quantification reagent was purchased from Molecular Probes Inc. (Eugene, OR). Seal restriction endonuclease was purchased from Pharmacia Biotech (Uppsala, Sweden). Micro B C A protein assay reagent kit was purchased from Pierce (Rockford, IL). p C M V C A T and pCMV(3gal plasmid were as outlined in sections 2.2.3 and 3.2.2. 4.2.2 Purification of plasmid of different conformations Linearized pCMVPgal was obtained by treating samples of pCMVPgal of mixed conformations with Seal restriction endonuclease at 37°C overnight. The resulting plasmid was purified by phenol/chloroform extraction, and then ethanol-precipitated after treatment with sodium acetate. Confirmation of linearized pCMVPgal (7164 bp) was done by 1 % agarose gel electrophoresis against a 1 kilobase ladder. To obtain supercoiled plasmid, purified pCMVPgal plasmid containing different conformations was further purified by cesium chloride (CsCl) gradient centrifugation (Sambrook et al., 1989). Briefly, a sample of plasmid (5 mg) was treated with 80 pl of ethidium bromide (10 mg/mL) in the dark for 20 min. The treated sample was then added to a CsCl (1.1 133 g/mL) gradient in T E buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA) in Beckman OptiSeal ultracentrifuge tubes. It was then centrifuged for 18 h at 60,000 rpm in a Beckman Optima XL-100K Ultracentrifuge (NVTi65 rotor). After centrifugation, two red bands containing DNA were observed. The top band containing mainly relaxed circular, dimer, concatamer or aggregates of plasmids was removed by needle suction. The bottom band containing supercoiled plasmid was isolated similarly, and was then repeatedly extracted with NaCl saturated isopropanol until a colorless solution was obtained. The bottom aqueous layer was then treated with 1 mL of 3 M sodium acetate, and the resulting plasmid was then ethanol-precipitated and washed. This procedure was repeated four times until the majority of the plasmid was identified to be supercoiled DNA as confirmed by 1 % agarose gel electrophoresis. To obtain relaxed circular plasmid, a sample of supercoiled DNA was incubated with Topoisomerase I in reaction buffer (50 mM Tris-HCl (pH 7.5), 50 mM KC1, 10 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM dithiothreitol, 30 Lig/mL bovine serum albumin) at 37°C for 2 days. The resulting mixture was then purified by CsCl gradient centrifugation. The upper band was isolated and purified as outlined above. The identity was confirmed by 1 % agarose gel electrophoresis. 4.2.3 Quantification of DNA entrapment by PicoGreen assay The quantification of SPLP formed with plasmid of different conformations was done using PicoGreen assay. Briefly, a column chromatography procedure employing D E A E -Sepharose CL-6B to remove free DNA was utilized. An aliquot of PicoGreen dye was diluted 2,000 fold in 150 mM NaCl, 20 mM HEPES buffer (pH 7.4) ("PicoGreen 134 buffer"). Fluorescence intensity was measured with an excitation wavelength of 480 nm and an emission of 520 nm. The intensity was zeroed with blank of the diluted PicoGreen buffer. A standard curve of fluorescence intensity of known pCMVPgal content (0 - 20 L i g ) was constructed in the presence of PicoGreen buffer. Samples of SPLP dialysate (0.5 uL) in 2 mL of PicoGreen buffer treated with 10 uL of 10% Triton X-100 were used as reference for the PicoGreen fluorescence. The remaining crude SPLP was applied to a DEAE-Sepharose column (diameter 1.0 cm, height 3.0 cm) and eluted using 150 mM NaCl, 20 mM HEPES (pH 7.4) buffer. The percentage of recovery was obtained by comparing the PicoGreen fluorescence intensity of an aliquot (4.0 uL) of the eluant in 2 mL PicoGreen buffer containing 10 uL of 10 % Triton X-100, with the reference fluorescence after volume adjustment. Thus, the % of recovery represents the fraction of DNA associated with the SPLP and sequestered from the anion exchanger, whereas the 1 4 C - C H E recovery represents the fraction of lipid in the void volume. Free plasmid D N A bound to the column could be washed out using 10 mL of a 5.0 M NaCl solution. Correlation studies using both radioactivity (section 3.2.5) and PicoGreen assay as methods for the quantification of DNA are shown to be comparable. The protocol for the purification of SPLP using sucrose density gradient centrifugation was described in section 3.2.6. Quantification of DNA was done using the PicoGreen assay as outlined above. The fluorescence intensity of an aliquot of 10 uL of purified SPLP treated with 10 uL of 10 % Triton X-100 in 2 mL of PicoGreen buffer was measured and compared to sample before sucrose density gradient after volume adjustment. The overall % recovery of DNA and lipid were computed by multiplying the % recovery from D E A E column with the % recovery from sucrose density gradient. 135 4.3 RESULTS 4.3.1 Influence of plasmid-to-lipid ratio on plasmid entrapment Previous work has shown that incubation of plasmid DNA with the lipid mixture DOPE, PEG2ooo-ceramide and the cationic lipid DODAC in the presence of detergent (OGP) followed by dialysis results in the formation of stabilized plasmid-lipid particles (SPLP) (Wheeler et al., 1998). However, the SPLP preparation procedure results in particles that contain plasmid and a large proportion of empty vesicles. As shown in Wheeler et al. (1998), these less dense empty vesicles can be separated from SPLP using sucrose density step gradient centrifugation; it would be advantageous, however, to reduce the proportion of empty vesicles in the SPLP preparation. One possibility is to increase the initial plasmid-to-lipid ratio in the dialysis medium. If the trapping efficiency is not proportionally reduced at higher plasmid-to-lipid ratios, fewer empty vesicles should be produced. Plasmid trapping efficiencies were therefore measured, employing the detergent dialysis protocol, for situations where the plasmid content was held constant at 25 pg p C M V C A T and the lipid content was varied over the range from 1 to 10 pmol. The lipid composition was DOPE/DODAC/PEG-CerCi 4 ; (82.5:7.5:10; molar ratios). As shown in Figure 4.1, the plasmid trapping efficiency increased almost linearly from 20 % to more than 50 % as the lipid content was increased from 1.0 to 5.0 pmol and plateaus thereafter. Thus decreases in the lipid content from 5 pmol total lipid resulted in proportionally reduced trapping efficiencies; as a result, the proportion of lipid in empty vesicles was not reduced. 136 Figure 4.1 Influence of total lipid content on DNA entrapment SPLP were prepared by the detergent dialysis procedure from 25 L i g p C M V C A T and varying amounts of total lipid (DOPE/DODAC/PEG-CerCi 4; 82.5:7.5:10; mol ratios). Recovery of 3 H - p C M V C A T (solid line) and the lipid marker 1 4 C - C H E (dotted line) are represented as the average and standard deviation from three individual experiments. 0 4 — . 1 . 1 . 1 . 1 . 1 0 2 4 6 8 10 Amount of lipid (umol) 137 An alternative method for reducing the empty vesicle fraction is to hold the lipid content constant and increase the plasmid concentration. Although this may appear to be formally equivalent to decreasing the lipid content and holding the plasmid concentration steady, significantly different trapping behaviour was observed. As demonstrated in Figure 4.2, plasmid trapping efficiencies of approximately 50 % were maintained at plasmid contents as high as 350 u.g, which corresponds to a plasmid-to-lipid ratio of 70 pg plasmid/umol lipid. If the plasmid-to-lipid ratio was increased to this value by decreasing the lipid content (Figure 4.1), the trapping efficiency was estimated to be approximately 10 %. 4.3.2 Effects of plasmid conformation on entrapment The detergent dialysis encapsulation process has been hypothesized to involve formation of hydrophobic plasmid DNA-cationic lipid intermediates (Wheeler et al., 1998). It would appear probable that penetration of the cationic lipid to certain plasmid nucleotide phosphates could be sterically inhibited for supercoiled plasmid as compared to linearized plasmid, for example, thus limiting the efficiency of encapsulation. In order to test this, supercoiled, relaxed circular and linearized plasmid DNA were prepared from the pCMV(3gal plasmid as described under section 4.2.2. Figure 4.3 depicts gel electrophoretic patterns of the isolated supercoiled (lane 3), relaxed circular (lane 4) and linearized (lane 5) pCMVPgal preparations. The unseparated pCMVPgal contains a mixture of conformations; composed mainly of relaxed circular, supercoiled, and dimer, concatamer or aggregates of plasmid (Figure 4.3, lane 2). 138 Figure 4.2 Influence of plasmid DNA content on plasmid DNA entrapment efficiency in SPLP SPLP were prepared by the detergent dialysis procedure from 5.0 pmol total lipids (DOPE/DODAC/PEG-CerCi 4; 82.5:7.5:10; mol ratios). Recovery of 3 H - p C M V C A T (solid line) and the lipid marker 1 4 C - C H E (dotted line) are represented as the average and standard deviation of three individual experiments. 100 80 0 1111111111111111111111111111111111111 0 100 200 300 Amount of plasmid (ug) 400 139 Figure 4.3 Agarose gel electrophoretic patterns of pCMVPgal plasmid of different conformations For each of the lanes, 0.5 u.g plasmid DNA was loaded onto the 1 % agarose gel. Lane 1 and 6 are the 1 kb DNA ladders. Lane 2 contains the unseparated pCMVPgal containing different conformations. Lane 3, 4, and 5 contains supercoiled, relaxed circular, and linearized pCMVPgal, respectively. The numbers on the right hand side of the panel indicate the size of the linear DNA standard. 140 The encapsulation properties of plasmid of different conformations in SPLP prepared from 5 pmol DOPE/DODAC/PEG-CerCg (72.5:7.5:20) were examined (Figure 4.4). At low plasmid-to-lipid ratios, all plasmid conformations exhibited high entrapment in SPLP. When greater than 50 pg plasmid was used, SPLP formed with linearized plasmid gave the best entrapment level of approximately 80 % (Figure 4.4C). For SPLP formed with supercoiled or relaxed circular plasmid, only 30 - 40 % and 40 - 50 % entrapment was observed, respectively (Figures 4.4A and B). The particle sizes of SPLP formed with different conformations were in the range of 120 - 150 nm diameter as measured by quasielastic light scattering (QELS) using particle mode. 4.3.3 SPLP formed from supercoiled plasmid give the highest in vitro transfection The in vitro transfection properties of purified SPLP formed with supercoiled, relaxed circular and linearized pCMVPgal were investigated employing BHK cells. As shown in Figure 4.5A, SPLP formed from supercoiled and relaxed circular plasmid induced higher levels of transfection than SPLP containing linearized plasmid. Studies on the transfection properties of DNA-cationic lipid complexes formed with pCMVpgal of different conformations were performed in parallel. As shown in Figure 4.5B, DNA-cationic lipid complexes formed with supercoiled pCMVPgal also gave higher transfection than complexes formed with linearized plasmid. 4.3.4 Stability of plasmid conformations in SPLP Plasmid DNA can undergo changes in conformation and organization depending on the environment experienced (Sambrook et al., 1989; Arscott et al., 1990; Bhattacharya and 141 Figure 4.4 Plasmid entrapment properties of SPLP containing pCMVPgal of different conformations SPLP composed of DOPE/DODAC/PEG-CerC 8 (72.5:7.5:20; mol ratios) were prepared from 5.0 umol total lipids in the presence of varying amounts of pCMVPgal in the supercoiled (A), relaxed circular (B) and linearized (C) conformations. The recovery of pCMVPgal from the D E A E Sepharose CL-6B column was quantified using the PicoGreen assay (solid line), whereas the lipid recovery was quantified by measuring 1 4 C - C H E (dotted line). The average and standard deviation from three individual experiments are shown. 100 0 100 200 300 400 Amount of plasmid (ug) 100 100 100 200 300 Amount of plasmid (ug) 400 100 200 300 Amount of plasmid (ug) 400 142 Figure 4.5 Comparison of the transfection ability of DNA-cationic lipid complexes and SPLP formed with different plasmid conformations The transfection activity resulting from (A) SPLP composed of DOPE/DOTMA/PEG-CerCg (73:7:20; mol ratios) and (B) pCMVpgal-DOTMA/DOPE (1:1) complexes using unseparated pCMVPgal (M), supercoiled (S), relaxed circular (R), and linearized (L) pCMVpgal are illustrated. SPLP containing 2.0 pg pCMVpgal and pCMVPgal-DOTMA/DOPE (1:1) complexes containing 0.5 pg pCMVpgal were employed using 24 h and 4 h transfection times, respectively. The average and standard deviation from triplicates are shown. M 300 M 143 Manual, 1997). Thus, efforts have been made to determine whether the encapsulated plasmid conformation in the SPLP is the original conformation. A 1 % agarose gel electrophoretic pattern of the SPLP formed with different plasmid conformations is shown in Figure 4.6. The unseparated, supercoiled, relaxed circular and linearized pCMVpgal (lanes 2 - 5 , respectively) are shown for reference. The profiles of plasmid from purified SPLP containing plasmids of different conformations are shown in lanes 6 to 9. It may be noted that plasmid entrapment in the SPLP appears to promote some conversion of the supercoiled plasmid into the relaxed circular conformation (lanes 6 and 7). In contrast, the conformation of relaxed circular or linearized plasmid is not affected by the encapsulation process (lanes 8 and 9). 144 Figure 4.6 Characterization of the influence of encapsulation on plasmid conformation by gel electrophoresis Free p C M V p g a l (lanes 2 to 5) and p C M V P g a l encapsulated in S P L P (lanes 6 to 9) was loaded onto a 1 % agarose gel. Lane 1 contains the 1 kilobase D N A ladder, and lane 2 contains the unseparated p C M V P g a l . Lanes 3, 4, and 5 contains isolated supercoiled, relaxed circular, and linearized p C M V P g a l , respectively. Lane 6 indicates the entrapped p C M V P g a l , while lanes 7, 8, and 9 illustrates the behaviour of plasmid in S P L P prepared from supercoiled, relaxed circular, and linearized p C M V p g a l , respectively. 1 2 3 4 5 6 7 8 9 145 4.4 DISCUSSION This investigation was focused on characterizing the influence of the plasmid component on entrapment and in vitro transfection properties of S P L P . The major findings concern the dependence of encapsulation on plasmid concentration and conformation, and the influence of plasmid conformation on transfection potency. Wi th regard to the influence of plasmid concentration on encapsulation efficiency, Wheeler et al. (1998) demonstrated that S P L P containing plasmid could be isolated by density centrifugation from empty vesicles formed during the dialysis process. It would be advantageous to improve the plasmid entrapment efficiency and minimize the formation of these empty vesicles. One obvious method is to increase the plasmid-to-lipid ratio in the S P L P formulation. Surprisingly, when the plasmid-to-lipid ratio is increased by decreasing the l ipid content during dialysis, the D N A trapping efficiency is decreased; whereas the trapping efficiency is increased i f the plasmid concentration is increased while keeping the l ipid concentration constant. This behaviour may be related to the importance of micelle surface charge for S P L P formation. A s shown here and elsewhere (Wheeler et al., 1998), for a fixed detergent concentration, encapsulation is sensitive to cationic l ipid content. Decreasing the l ipid content increases the detergent-to-lipid ratio and thus dilutes the cationic l ipid, possibly reducing the micelle-plasmid association, which initiates S P L P formation. The relative independence of plasmid encapsulation efficiency from an increase of the plasmid-to-lipid ratio over the range 0 - 7 0 iig/umol (Figure 4.2) provides a direct method for decreasing the proportion of empty vesicles in the S P L P preparation 146 following detergent dialysis. This observation is also consistent with the model of S P L P formation proposed by Wheeler et al. (1998). Briefly, this model suggests that S P L P form via the condensation of cationic lipid-containing micelles around a single plasmid. A s a result, S P L P should continue to form as the plasmid content is increased as long as there is an excess of micelles. This model is also consistent with the observation that the encapsulation efficiency appears to be significantly reduced at plasmid-to-lipid ratios of 70 pg/pmol l ipid and higher. A s discussed by Wheeler et al. (1998), S P L P containing a single 5 kb plasmid exhibit a plasmid-to-lipid ratio of approximately 62.5 pg/pmol, and thus the reduced efficiency may be attributed to depletion of the micellar pool at the high plasmid concentrations. A remaining puzzle concerns the fact that encapsulation efficiency is 50 - 60 % even when the micellar lipids are in excess. It would appear that a certain fraction of the plasmid is not available for encapsulation by the detergent dialysis procedure. The conformation of the plasmid could have a significant influence on the entrapment properties of S P L P . For example, it is likely that some proportion of the nucleotide phosphates of plasmids in the supercoiled conformation is not able to pair with a cationic l ipid due to steric exclusion effects, reducing encapsulation. This is consistent with the 40 % encapsulation levels noted for supercoiled plasmid as compared to ~ 80 % for linearized plasmid. However, the fact that relaxed circular and supercoiled plasmid exhibit approximately equal encapsulation efficiencies suggests that other conformational effects may also play a role in limiting encapsulation efficiency. The results presented here indicate that, on the basis of encapsulation efficiency, S P L P formed from linearized plasmid offer substantially improved properties as 147 compared to supercoiled plasmid. It is therefore unfortunate that the linearized plasmid employed here exhibits such poor transfection properties. Several possible factors may contribute to these phenomena. First, adequate expression of the P-gal gene carried by the plasmid D N A requires proper transcription and translation processes. If the plasmid D N A is linearized while keeping an intact P-gal gene as in this case, then certain transcription elements such as the upstream or downstream enhancer regions of the plasmid may not be situated at the appropriate site. Another possible reason may be attributed to the arrangement of linearized plasmid entrapped in S P L P . I f these linearized plasmids can bind more cationic l ipid through the fully accessible anionic nucleotide phosphates, then release of plasmid from the cationic lipids into the cell cytoplasm may be inhibited. Similar rationales can also be used to explain the phenomena of superior transfection behavior for plasmid DNA-cationic l ipid complexes formed with the supercoiled plasmid as compared to that of linearized plasmid (Figure 4.5B). This study characterizes the influence of plasmid conformation and concentration on D N A entrapment and in vitro transfection properties in S P L P . It is demonstrated that high encapsulation efficiencies can be achieved employing linearized plasmid D N A , however S P L P containing the linearized plasmid exhibit much reduced transfection potency. S P L P containing supercoiled plasmid D N A in lipid-based carrier systems give superior in vitro transfection properties. 148 CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS 5.1 SUMMARY The studies presented in this thesis were aimed at characterizing and comparing two cationic lipid-based DNA delivery systems. First, the structural and fusogenic properties of plasmid DNA-cationic lipid complexes were investigated and related to the transfection properties. Then factors influencing the formation and transfection properties of the small stabilized plasmid-lipid particles were characterized and compared with the larger complexes. The experiments described in Chapter 2 were designed to examine the structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA. First, the ability of D O T M A to stabilize the hexagonal-forming DOPE into a bilayer organization was analyzed using 3 1 P NMR. It was shown that cationic vesicles composed of equimolar D O T M A and DOPE can be formed as the system is stabilized in a bilayer organization. As indicated by fusion assays and freeze fracture E M , addition of pCMV5 plasmid induces aggregation and fusion between these cationic vesicles to form DNA-liposome aggregates and/or DNA-lipid assemblies. Interestingly, the phospholipids in these large lipid assemblies exhibit an isotropic 3 1 P NMR resonance, which indicates rapid isotropic motion of the phospholipid. Complexes formed with DOPC exhibit only a broad bilayer signal. The observation of structures resembling "lipidic particles" in freeze fracture E M suggests the isotropic signal in 3 1 P NMR may be attributed to fusion intermediates present in complexes formed with DOTMA/DOPE, but not with DOTMA/DOPC. Next, the fusogenic ability of complexes formed with DOTMA/DOPE and DOTMA/DOPC 149 L U V s were correlated with in vitro transfection. D O T M A / D O P E complexes were found to have superior in vitro transfection potential compared to complexes formed with D O T M A / D O P C L U V s . It was also shown that complexes formed with the hexagonal-forming D O P E have higher fusogenic ability with anionic D O P S / D O P C vesicles. The complexes exhibited superior transfection and fusogenic tendencies when formed at a positive-to-negative charge ratio of 1.0, and could also be isolated in high yield in the pellet fraction following centrifugation at this ratio. The results presented here suggest that non-bilayer l ipid structures could play a direct role in destabilizing the bilayer membrane and promoting membrane fusion processes, thus facilitating the transfection event. The investigation in Chapter 3 was designed to examine the factors that influence the formation of stabilized plasmid-lipid particles (SPLP). S P L P are about 100 nm in diameter, have the potential of displaying long circulation lifetimes in vivo, and protect entrapped plasmid against serum or DNase degradation. First, the composition of the cationic lipids and the type of cationic l ipid used in S P L P were compared in terms o f plasmid entrapment and in vitro transfection levels. Although a slight variation in the cationic l ipid content is observed in forming S P L P using different cationic lipids, similar plasmid entrapment levels were obtained. It was observed that S P L P formed with glycerol-based cationic lipids ( D O D M A - A N and D O T M A ) have a high transfection potency compared to other cationic lipids ( D O D A C , D O S D A C and D C - C H O L ) . Next, the influence of PEG-Cer on the formation and transfection of these particles was investigated. It was demonstrated that the large P E G polymer can substantially influence the formation of S P L P by lowering the plasmid entrapment and increasing the proportion 150 of cationic lipid in the lipid formulation. In addition, SPLP formed with large PEG polymers (PEG5000 or 2000) inhibit the cell association process and lower the transfection potency of these particles. Since the presence of PEG inhibits liposome clearance, it is important to retain PEG-Cer within the particles such that a circulation lifetime is obtained which would allow particle accumulation at the target cells. On the other hand, the release of PEG-Cer at the target site is necessary if uptake and transfection are to occur. One way to balance these two requirements is to vary the anchor chain length of PEG-Cer, as longer side chains have more hydrophobic interaction with the bilayer and will remain in the particles for a longer time. It was shown that SPLP formed with a shorter acyl chain anchor (PEG-CerCs) require a larger PEG content in the lipid formulation, and exhibit superior in vitro transfection than do SPLP formed with PEG-CerCi4 or C2o- Finally, the high transfection abilities of the conventional cationic lipid complexes were compared and correlated with their uptake by cells. Due to the higher net positive charge density, DNA-cationic lipid complexes demonstrate higher transfection abilities than SPLP. However, the plasmid delivered by the complexes is partially degraded during the transfection process. The experiments in Chapter 4 examined the influence of the plasmid component in SPLP on DNA entrapment and transfection properties. First, the plasmid-to-lipid ratio was optimized to yield SPLP exhibiting high DNA entrapment with minimal amount of lipid. Then, the influence of plasmid conformations in SPLP was analyzed. Although SPLP formed with supercoiled or relaxed circular plasmid have lower plasmid entrapment levels, these systems exhibit superior transfection abilities over SPLP formed with linearized plasmid. 151 Despite lower in vitro transfection levels, S P L P have three advantages over plasmid DNA-cat ionic l ipid complexes as in vivo D N A carriers. First, the small size of about 100 nm and P E G coating provides the basis for long circulation times for in vivo applications, facilitating accumulation at disease sites such as tumors. Second, the ability of these particles to protect encapsulated plasmid D N A provides a foundation for efficient plasmid delivery. Finally, the dissociation of the PEG-Cer from the S P L P after arrival at the disease site offers the potential for association with target cells. 5.2 Future Directions In Chapter 2, the structural and fusogenic properties of the conventional plasmid D N A -cationic l ip id complexes were correlated to transfection properties. Although the phospholipid phase behavior in cationic l ipid systems in the presence of D N A were analyzed, it would be of interest to pursue further research on the behavior of the cationic l ipid, D O T M A , in the presence of plasmid D N A to determine whether the D O T M A is segregated into local domains following interaction with plasmid. This could be done by utilizing 2H-labeled D O T M A for analysis by 2 H N M R . Furthermore, since DNA-cat ionic l ipid complexes are heterogenous, it would be of interest to fractionate the complexes by a density gradient, and analyze each fraction using Q E L S , N M R , and freeze fracture E M to reveal other possible intermediate structures. This could then be correlated with appropriate transfection studies. A requirement for successful gene therapy is the development of an efficient D N A delivery system for in vivo applications. Thus, in Chapters 3 and 4, we focused on analyzing the small S P L P system, which is designed for in vivo gene delivery. Further work is still required to enhance the plasmid entrapment and transfection potency of these 152 systems. As indicated in Chapter 3, a major source limiting the transfection ability of SPLP is the lower cellular uptake of the particles. This can be attributed to the low cationic lipid content in SPLP as compared to that in the complexes. 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